Inhibitors of lysyl oxidases

ABSTRACT

Described herein are novel compounds that block the activity of LOX family members having good IC50 values, no cellular toxicity below 10 μM, induce sensitization of the cells to doxorubicin, strong activity in a recombinant LOX/LOXL2 activity, and a chemical structure that is drug-like and does not have a PAINS flag, as well as, methods of treatment using the compounds with respect to cancer, organ fibrosis, neurodegenerative and cardiovascular diseases.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under NationalInstitutes of Health grant number P20 GM109091 and National Institutesof Health grant number RO1 CA267101. The government has certain rightsin the invention.

TECHNICAL FIELD

The subject matter disclosed herein is generally directed topharmaceutically acceptable compositions, and pharmaceuticallyacceptable salts thereof, and treatment regimens for inhibitions of LOXor LOX family activity wherein the compounds of the present disclosureare useful for treating a variety of diseases, disorders, or conditions,associated with LOX activity and the compounds are also useful for thestudy of LOX enzymes in biological and pathological phenomena.

BACKGROUND

Lysyl oxidase (LOX) family are copper-dependent amine oxidases. Thereare five (5) different members of the LOX family proteins (LOX, LOXL1,LOXL2, LOXL3 and LOXL4) that share a high degree of homology at thecatalytic carboxy terminal end. See, Barker, H. E., Cox, T. R. & Erler,J. T. The rationale for targeting the LOX family in cancer. Nat RevCancer 12, 540-552, doi:10.1038/nrc3319 (2012). Importantly, LOX andLOXL1 are synthesized as propeptides and cleaved by bone morphogenicprotein 1 (BMP1) metalloprotease to obtain functional enzymes. See,Trackman, P. C., Bedell-Hogan, D., Tang, J. & Kagan, H.Post-translational glycosylation and proteolytic processing of a lysyloxidase precursor. Journal Of Biological Chemistry 267, 8666-8671(1992). LOX family of proteins mediate conversion of lysine residues incollagen I and elastin precursors into highly reactive aldehydes;thereby triggering crosslinking and stabilization of ECM proteins thatregulate cell adhesion, motility, and invasion. See, Kagan, H. M. & Li,W. Lysyl oxidase: properties, specificity, and biological roles insideand outside of the cell. Journal of Cellular Biochemistry 88, 660-672,doi:10.1002/jcb.10413 (2003). LOX family members play key roles duringembryogenesis and organ development. See, Maki, J. M. et al. Lysyloxidase is essential for normal development and function of therespiratory system and for the integrity of elastic and collagen fibersin various tissues. The American Journal of Pathology 167, 927-936(2005), Brody, J. S., Kagan, H. & Manalo, A. Lung lysyl oxidaseactivity: relation to lung growth. American Review of RespiratoryDisease 120, 1289-1295 (1979), and Wei, S., Gao, L., Wu, C., Qin, F. &Yuan, J. Role of the lysyl oxidase family in organ development.Experimental and Therapeutic Medicine 20, 163-172 (2020). While all theLOX family members have a wide distribution in organs; all of them areexpressed in the heart and colon. See, Yang, N., Cao, D.-F., Yin, X.-X.,Zhou, H.-H. & Mao, X.-Y. Lysyl oxidases: emerging biomarkers andtherapeutic targets for various diseases. Biomedicine & Pharmacotherapy131, 110791 (2020), Martins, R. P., Leach, R. E. & Krawetz, S. A.Whole-body gene expression by data mining. Genomics 72, 34-42 (2001),Jourdan-Le Saux, C., Tomsche, A., Ujfalusi, A., Jia, L. & Csiszar, K.Central nervous system, uterus, heart, and leukocyte expression of theLOXL3 gene, encoding a novel lysyl oxidase-like protein. Genomics 74,211-218 (2001), Asuncion, L. et al. A novel human lysyl oxidase-likegene (LOXL4) on chromosome 10q24 has an altered scavenger receptorcysteine rich domain. Matrix Biology 20, 487-491 (2001), Kim, Y., Boyd,C. D. & Csiszar, K. A New Gene with Sequence and Structural Similarityto the Gene Encoding Human Lysyl Oxidase (*). Journal of BiologicalChemistry 270, 7176-7182 (1995), and Maki, J. M., Tikkanen, H. &Kivirikko, K. I. Cloning and characterization of a fifth human lysyloxidase isoenzyme: the third member of the lysyl oxidase-relatedsubfamily with four scavenger receptor cysteine-rich domains. Matrixbiology 20, 493-496 (2001). Importantly, all members have so far beenassociated with cancer, particularly with metastasis and recently withchemoresistance, with LOX (first identified member) and LOXL2 being themost intensely studied members of the family See, Xiao, Q. & Ge, G.Lysyl oxidase, extracellular matrix remodeling and cancer metastasis.Cancer Microenviron 5, 261-273, doi: 10.1007/s 12307-012-0105-z (2012).

The inventors have reported that the hypoxia-induced LOX expression is adriver of TNBC chemoresistance. See, Saatci, O. et al. Targeting lysyloxidase (LOX) overcomes chemotherapy resistance in triple negativebreast cancer. Nat Commun 11, 2416, doi:10.1038/s41467-020-16199-4(2020). The inventors demonstrated that LOX not only enhances ECMcrosslinking and generates a barrier to chemotherapy, but also fuelscell proliferation and therapy resistance via activating pro-survivalintegrin signaling in TNBC. See, Id. Inhibition of LOX activity viaβ-aminopropionitrile (BAPN, a LOX family inhibitor) or shRNA-mediatedknockdown of LOX reduced collagen cross-linking, leading to accumulationof doxorubicin in TNBC cells, reduced FAK/Src signaling and led tochemosensitization both in vitro and in vivo. In gastric cancer, LOXexpression has been shown to be increased, and it is correlated with theinvasion depth and lymph node metastasis while inhibition of LOX viasiRNA suppressed the hypoxia-induced EMT of gastric cancer cells. See,Kasashima, H. et al. Lysyl oxidase-like 2 (LOXL2) from stromalfibroblasts stimulates the progression of gastric cancer. Cancer Letters354, 438-446 (2014). High LOX expression is also associated with EMTmarkers and predicts early recurrence and poor survival in HCC patients.See, Umezaki, N. et al. Lysyl oxidase induces epithelial-mesenchymaltransition and predicts intrahepatic metastasis of hepatocellularcarcinoma. Cancer science 110, 2033-2043 (2019). Another study showedthat LOX expression is required for the mutant p53 driven invasion inpancreatic cancer, and combination of gemcitabine with LOX inhibitionled to the tumor necrosis. See, Miller, B. W. et al. Targeting theLOX/hypoxia axis reverses many of the features that make pancreaticcancer deadly: inhibition of LOX abrogates metastasis and enhances drugefficacy. EMBO Molecular Medicine 7, 1063-1076 (2015). Furthermore, LOXmRNA is increased in human clear cell renal cell carcinoma (ccRC), andthe ectopic expression of LOX resulted in migration and collagenstiffness. See, Di Stefano, V. et al. Major action of endogenous lysyloxidase in clear cell renal cell carcinoma progression and collagenstiffness revealed by primary cell cultures. The American Journal ofPathology 186, 2473-2485 (2016) and Takahashi, M. et al. Gene expressionprofiling of clear cell renal cell carcinoma: gene identification andprognostic classification. Proceedings of the National Academy ofSciences 98, 9754-9759 (2001).

Among the other members of the LOX family, LOXL1 is the least studiedmember in the cancer context. A few studies showed its oncogenicpotential in different cancers. Elevated expression of LOXL1 was foundin primary lung cancer tissues, and LOXL1-overexpression in mice led togreater number of lung metastatic nodules. See, Lee, G.-H. et al. Lysyloxidase-like-1 enhances lung metastasis when lactate accumulation andmonocarboxylate transporter expression are involved. Oncology Letters 2,831-838 (2011). In pancreatic cancer patients, LOXL1 mRNA was shown tobe increased when compared to normal pancreatic tissue. See, Le Calve,B. et al. Lysyl oxidase family activity promotes resistance ofpancreatic ductal adenocarcinoma to chemotherapy by limiting theintratumoral anticancer drug distribution. Oncotarget 7, 32100 (2016).

The role of LOXL2 is extensively studied in cancer and fibrosis. Inbreast cancer, high expression of both LOX and LOXL2 shows a strongcorrelation with invasive potential of metastatic mammary tumors. See,Kirschmann, D. A. et al. A molecular role for lysyl oxidase in breastcancer invasion. Cancer Research 62, 4478-4483 (2002). A high level ofLOXL2 expression was also detected in HCC cells and tissues that isassociated with the poor disease-free survival and overall survival ofHCC patients. See, Ninomiya, G. et al. Significance of Lysyloxidase-like 2 gene expression on the epithelial-mesenchymal status ofhepatocellular carcinoma. Oncology Reports 39, 2664-2672 (2018). Higherexpression of LOXL2 enhances the invasion ability of pancreatic cancercells, and a higher distant recurrent rate was found in LOXL2-positivetumors in pancreatic cancer patients. See, Park, J. S. et al. Emergingrole of LOXL2 in the promotion of pancreas cancer metastasis. Oncotarget7, 42539 (2016). In primary gastric tumors, higher LOXL2 expression iscorrelated with higher tumor invasion, higher lymph node metastasis andpoorer overall patient survival. See, Peng, L. et al. Secreted LOXL2 isa novel therapeutic target that promotes gastric cancer metastasis viathe Src/FAK pathway. Carcinogenesis 30, 1660-1669 (2009).

LOXL3 overexpression is observed in gastric cancer, and the oncogenicrole of LOXL3 with respect to increasing invasion has been shown ingastric cancer. See, Kasashima, supra. In melanoma, LOXL3 regulatesgenome integrity by binding several key members of the DNA damageresponse and promotes tumor growth in vivo. See, Santamaria, P. G. etal. Lysyl oxidase-like 3 is required for melanoma cell survival bymaintaining genomic stability. Cell Death & Differentiation 25, 935-950(2018). In addition to the gastric cancer and melanoma, LOXL3 expressionwas also upregulated in breast cancer, see Jeong, Y. J. et al.Association between lysyl oxidase and fibrotic focus in relation withinflammation in breast cancer. Oncology Letters 15, 2431-2440 (2018),myeloproliferative neoplasm, see Tadmor, T. et al. The expression oflysyl-oxidase gene family members in myeloproliferative neoplasms.American Journal of Hematology 88, 355-358 (2013), and ovariancarcinoma. See, Dufresne, J. et al. The plasma peptides of ovariancancer. Clinical Proteomics 15, 1-19 (2018). High LOXL4 expression isassociated with the poor prognosis of HCC patients, and it induces theintrahepatic and pulmonary metastasis of HCC in in vivo. See, Li, R. etal. Exosome-mediated secretion of LOXL4 promotes hepatocellularcarcinoma cell invasion and metastasis. Molecular cancer 18, 1-19(2019). LOXL4 overexpression is associated with poor overall survival inbreast cancer patients, see Choi, S. K., Kim, H. S., Jin, T. & Moon, W.K. LOXL4 knockdown enhances tumor growth and lung metastasis throughcollagen-dependent extracellular matrix changes in triple-negativebreast cancer. Oncotarget 8, 11977 (2017), and LOXL4 inhibitioninhibited the migration and invasion of lung adenocarcinoma cells. See,Xie, S., Liu, G., Huang, J., Hu, H. B. & Jiang, W. miR-210 promotes lungadenocarcinoma proliferation, migration, and invasion by targeting lysyloxidase-like 4. Journal of Cellular Physiology 234, 14050-14057 (2019).Another study in head and neck squamous cell carcinomas (HNSCC) showedthat high LOXL4 expression was correlated with tumor stage and lymphnode metastasis. See, Weise, J. B. et al. LOXL4 is a selectivelyexpressed candidate diagnostic antigen in head and neck cancer. Europeanjournal of cancer 44, 1323-1331 (2008).

Human fibrotic diseases are composed of idiopathic pulmonary fibrosis,liver fibrosis, cardiovascular fibrosis and renal fibrosis that sharethe common feature of excessive deposition of ECM fibrillar proteins,such as collagen in damaged tissues, leading to disrupted ECMhomeostasis and organ failure. See, Kim, Y.-M., Kim, E.-C. & Kim, Y. Thehuman lysyl oxidase-like 2 protein functions as an amine oxidase towardcollagen and elastin. Molecular Biology Reports 38, 145-149 (2011),Chen, L., Li, S. & Li, W. LOX/LOXL in pulmonary fibrosis: potentialtherapeutic targets. Journal of Drug Targeting 27, 790-796 (2019), Yang,J. et al. Targeting LOXL2 for cardiac interstitial fibrosis and heartfailure treatment. Nature Communications 7, 1-15 (2016), Chien, J. W. etal. Serum lysyl oxidase-like 2 levels and idiopathic pulmonary fibrosisdisease progression. European Respiratory Journal 43, 1430-1438 (2014),Steppan, J. et al. Lysyl oxidase-like 2 depletion is protective inage-associated vascular stiffening. American Journal of Physiology-Heartand Circulatory Physiology 317, H49-H59 (2019), Nave, A. H. et al. Lysyloxidases play a causal role in vascular remodeling in clinical andexperimental pulmonary arterial hypertension. Arteriosclerosis,Thrombosis, and Vascular Biology 34, 1446-1458 (2014), andMartinez-Revelles, S. et al. Lysyl oxidase induces vascular oxidativestress and contributes to arterial stiffness and abnormal elastinstructure in hypertension: role of p38MAPK. Antioxidants & RedoxSignaling 27, 379-397 (2017). In addition to role of the LOX familymembers in cancer, their upregulation and increased activity havefundamental roles in fibrosis. For example, the expression of both LOXand LOXL1 is increased upon TGF-β stimulation and they promotefibrogenesis in hepatic stellate cell and cardiac fibroblast. See, Lu,M., Qin, Q., Yao, J., Sun, L. & Qin, X. Induction of LOX byTGF-β1/Smad/AP-1 signaling aggravates rat myocardial fibrosis and heartfailure. IUBMB Life 71, 1729-1739 (2019) and Ma, L. et al. Knockdown ofLOXL1 inhibits TGF-β1-induced proliferation and fibrogenesis of hepaticstellate cells by inhibition of Smad2/3 phosphorylation. Biomedicine &Pharmacotherapy 107, 1728-1735 (2018). Furthermore, LOX expression isincreased in myocardial fibrosis which directly contributes to adversemyocardial remodeling in cardiac diseases. See, Spurney, C. F. et al.Dystrophin-deficient cardiomyopathy in mouse: expression of Nox4 and Loxare associated with fibrosis and altered functional parameters in theheart. Neuromuscular Disorders 18, 371-381 (2008). In physiologicalconditions, LOX family members contribute to the integrity andstabilization of a healthy vessel wall. See, Kumari, S., Panda, T. K. &Pradhan, T. Lysyl oxidase: its diversity in health and diseases. IndianJournal of Clinical Biochemistry 32, 134-141 (2017). However, earlyinduction and overexpression of LOX led to arterial stiffening inHutchinson-Gilford Progeria Syndrome. Furthermore, LOX is stronglyupregulated in young mice and inhibition of LOX led to improved arterialmechanics and cardiac function. See, von Kleeck, R. et al. Arterialstiffness in Hutchinson-Gilford Progeria Syndrome corrected byinhibition of Lysyl Oxidase. Moreover, both cardiac hypertrophy anddisfunction are associated with LOX overexpression which may be targetedfor the clinical management of hypertrophy and heart failure. See,Galán, M. et al. Lysyl oxidase overexpression accelerates cardiacremodeling and aggravates angiotensin II—induced hypertrophy. The FASEBJournal 31, 3787-3799 (2017). Overall, these studies show the key rolesof LOX and other LOX family members in fibrosis.

In addition to the role of LOX family members in cancer and fibroticdiseases, aberrant lysyl oxidase activity has also been shown in otherpathological conditions, including diabetes, neurodegeneration andadipose tissue dysfunction. See, Wilhelmus, M. M., Bol, J. G., vanDuinen, S. G. & Drukarch, B. Extracellular matrix modulator lysyloxidase colocalizes with amyloid-beta pathology in Alzheimer's diseaseand hereditary cerebral hemorrhage with amyloidosis—Dutch type.Experimental Gerontology 48, 109-114 (2013) and Pastel, E. et al. Lysyloxidase and adipose tissue dysfunction. Metabolism 78, 118-127 (2018).For example, LOX overexpression is induced by hypoxia and inflammationin adipose tissue with obesity. See, Pastel, supra. In addition LOXexpression is induced in diabetes and led to apoptosis in rat retinalendothelial cells while siRNA knockdown of LOX reduced diabetes-inducedLOX expression and prevented vascular leakage associated with diabeticretinopathy. See, Chronopoulos, A., Tang, A., Beglova, E., Trackman, P.C. & Roy, S. High glucose increases lysyl oxidase expression andactivity in retinal endothelial cells: mechanism for compromisedextracellular matrix barrier function. Diabetes 59, 3159-3166 (2010).High LOX and LOXL2 level were observed in choroid neovascularization andthe inhibition of LOX and LOXL2 through neutralizing anti-LOX and LOXL2antibodies showed the functional contribution of LOX and LOXL2 indiabetic retinopathy. See, Roy, S., Ha, J., Trudeau, K. & Beglova, E.Vascular basement membrane thickening in diabetic retinopathy. Currenteye research 35, 1045-1056 (2010). Under physiological conditions, theLOX expression is low in rat brain; however, it accumulates in thelesion after brain injury. See, Gilad, G. M., Kagan, H. M. & Gilad, V.H. Lysyl oxidase, the extracellular matrix-forming enzyme, in rat braininjury sites. Neuroscience letters 310, 45-48 (2001). Interestingly,high LOX activity is observed during the plaque formation in Alzheimer'sdisease (AD) through ECM modulation. See, Gilad, G. M., Kagan, H. M. &Gilad, V. H. Evidence for increased lysyl oxidase, the extracellularmatrix-forming enzyme, in Alzheimer's disease brain. NeuroscienceLetters 376, 210-214 (2005).

Given the multifaceted functions of the LOX family members in fibrosis,cancer and other diseases, there is huge interest to identify anddevelop potent and tolerable LOX or LOX family inhibitors. BAPN is theearliest and most commonly used research tool to inhibit LOX; itinhibits all LOX family members and showed toxicity in the clinic. See,Keiser, H. R. & Sjoerdsma, A. Studies on beta-aminopropionitrile inpatients with scleroderma. Clin Pharmacol Ther 8, 593-602,doi:10.1002/cpt196784593 (1967) and Spencer, P. & Schaumburg, H.Lathyrism: a neurotoxic disease. Neurobehavioral toxicology andteratology 5, 625-629 (1983). Simtuzumab, a humanized monoclonalantibody binding LOXL2, was tested for patients with fibrosis in threedifferent clinical trials; however, these studies either terminated dueto lack of efficacy (NCT01769196) or resulted in no benefit (NCT01672866and NCT01672879). Furthermore, no clinical benefit has been reportedwith Simtuzumab in combination with chemotherapy in the first andsecond-line settings in pancreatic and colorectal adenocarcinomapatients, respectively. See, Benson, A. B., 3rd et al. A Phase IIRandomized, Double-Blind, Placebo-Controlled Study of Simtuzumab orPlacebo in Combination with Gemcitabine for the First-Line Treatment ofPancreatic Adenocarcinoma. Oncologist 22, 241-e215,doi:10.1634/theoncologist.2017-0024 (2017) and Hecht, J. R. et al. APhase II, Randomized, Double-Blind, Placebo-Controlled Study ofSimtuzumab in Combination with FOLFIRI for the Second-Line Treatment ofMetastatic KRAS Mutant Colorectal Adenocarcinoma. Oncologist 22,243-e223, doi:10.1634/theoncologist.2016-0479 (2017). More recently, aphenoxypyricline-containing small molecule, PAT-1251, an inhibitor ofLOXL2/LOXL3, showed promising results as an anti-fibrotic agent inpreclinical models, see Rowbottom, M. W. et al. Identification of4-(Aminomethyl)-6-(trifluoromethyl)-2-(phenoxy)pyridine Derivatives asPotent, Selective, and Orally Efficacious Inhibitors of theCopper-Dependent Amine Oxidase, Lysyl Oxidase-Like 2 (LOXL2). J Med Chem60, 4403-4423, doi:10.1021/acs.jmedchem.7b00345 (2017), and it iscurrently in Phase IIa clinical trial for myelofibrosis (NCT04679870).The haloallylamine-based small molecule inhibitors, PXS-S1A and PXS-S2Awere potent against LOX/LOXL2 and LOXL2, respectively, and the formerone inhibited lung and liver metastasis. See, Chang, J. et al.Pre-clinical evaluation of small molecule LOXL2 inhibitors in breastcancer. Oncotarget 8, 26066-26078, doi:10.18632/oncotarget.15257 (2017).An orally bioavailable aminomethylenethiophene (AMT)-based inhibitor,CCT365623 is active against LOX/LOXL2 and led to significant reductionin metastasis in a breast cancer animal model. See, Leung, L. et al.Anti-metastatic Inhibitors of Lysyl Oxidase (LOX): Design andStructure-Activity Relationships. J Med Chem 62, 5863-5884,doi:10.1021/acs.jmedchem.9b00335 (2019). A pan-lysyl oxidase inhibitorPXS-5505 was well-tolerated in healthy volunteers and is now beingtested in patients with primary, postpolycythernia vera (amyeloproliferative neoplasm) or post-essential thrombocythemiamyelofibrosis. See, How, J. et al. Evaluation of a Pan-Lysyl OxidaseInhibitor, Pxs-5505, in Myelofibrosis: A Phase I, Randomized, PlaceboControlled Double Blind Study in Healthy Adults. Blood 136,doi:10.1182/blood-2020-139223 (2020). Recently, the FDA cleared the testof PXS-5505 in combination with a PD-L1 inhibitor and an anti-VEGF drugas first line therapy in newly diagnosed patients with unresectable HCCcarcinoma (NCT05109052). In addition, Cyclosporin (CsA) is one of themost commonly used drugs after the solid organ transplantation and alsoit is the main cause of uremia. Importantly, combinatorial treatmentwith CsA and Pan-LOX inhibitor, PXS-5505 attenuated uremia andprogressive nephropathy. See, Nguyen, L. T. et al. Lysyl oxidaseinhibitors attenuate cyclosporin A-induced nephropathy in mouse.Scientific reports 11, 1-12 (2021). Despite the presence of severalon-going clinical trials using LOX family inhibitors in severaldiseases, there is still no LOX inhibitor approved by the FDA, showingthe necessity of developing novel, efficacious and tolerable inhibitorsagainst LOX or LOX family members.

According to the American Cancer Society, there will be an estimated 1.8million new cancer cases diagnosed and 606,520 cancer deaths in theUnited States. This number may be multiplied by at least eight toestimate the world cancer incidence/death numbers. Heart diseases arethe leading cause of death followed by cancer. Neurodegenerativediseases and fibrosis are also very common. Cancer is worldwide problem,and different therapy options are available e.g., chemotherapy,immunotherapy and an antibody-drug conjugate are available for patients.See www.cancer.org.

Accordingly, it is an object of the present disclosure to provide novelcompounds that block the activity of lysyl oxidases family members, witha potential to be developed further as cancer drugs to inhibitresistance to conventional therapies and metastasis, or as drugs totreat fibrotic disease.

Citation or identification of any document in this application is not anadmission that such a document is available as prior art to the presentdisclosure.

SUMMARY

The above objectives are accomplished according to the presentdisclosure by providing a method for inhibiting lysyl oxidases. Themethod may include administering a therapeutically effective amount of acompound or its pharmaceutically acceptable salt having formula I;

wherein: R1 is CH₃, R₂ is H, NH₂ and NHR, R₃ is H or CH₃, R₄ is H, R₅ isH, R₆ is NO₂, C₂OH, NHCOCH₃, or OH, R₇ is H, Q1 is N, Q2 is S, Q3 is N,Q4 is CH, Q5 is S, Q6 is CH, Q7 is CH, Q8 is C; and Q9 is CH wherein thecompound or its pharmaceutically acceptable salt inhibits cell-basedlysyl oxidase activity and/or recombinant protein based lysyl oxidaseactivity assays.

Further, the therapeutically effective amount of the compound or itspharmaceutically acceptable salt may be administered to treat aneurodegenerative disease, an angiogenesis-related disease, Alzheimer'sdisease, fibrosis including: liver fibrosis, pulmonary fibrosis, renalfibrosis, myocardial fibrosis, cystic fibrosis, idiopathic pulmonaryfibrosis, radiation-induced fibrosis, ocular fibrosis, Peylony's diseaseand scleroderma, or respiratory disorders, abnormal wound healing andrepair, postoperative surgery, cardiac arrest-related fibrosis, excessor abnormal deposition of fibrotic material, all associated withdisorders such as Crohn's disease and inflammatory bowel disease, liver,kidney and pancreas fibrosis, diabetes, cerebral hemorrhage withamyloidosis, cardiac hypertrophy, Hutchinson-Gilford Progeria Syndrome,retinopathy, chemoresistance, and/or a kidney disorder including: kidneyfibrosis, renal fibrosis, acute kidney injury, chronic kidney disease,diabetic nephropathy, glomerulosclerosis, vesicoureteral reflux,tubulointerstitial renal fibrosis and/or glomerulonephritis. Stillfurther, the cancer may be selected from the group comprising lungcancer; breast cancer; colorectal cancer; anal cancer; pancreaticcancer; prostate cancer; ovarian carcinoma; liver and bile ductcarcinoma; esophageal carcinoma; non-Hodgkin's lymphoma; bladdercarcinoma; carcinoma of the uterus; glioma, glioblastoma,medullablastoma, and other tumors of the brain; myelofibrosis, kidneycancer; cancer of the head and neck; cancer of the stomach; multiplemyeloma; testicular cancer; germ cell tumor; neuroendocrine tumor;cervical cancer; oral cancer, carcinoids of the gastrointestinal tract,breast, and other organs; signet ring cell carcinoma; mesenchymal tumorsincluding sarcomas, fibrosarcomas, haemangioma, angiomatosis,haemangiopericytoma, pseudoangiomatous stromal hyperplasia,myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumour,lipoma, angiolipoma, granular cell tumour, neurofibroma, schwannoma,angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyomaand/or a leiomysarcoma. Again, the compound or its pharmaceuticallyacceptable salt may have formula:

Moreover, the compound or its pharmaceutically acceptable salt may haveformula:

Indeed, the compound or its pharmaceutically acceptable salt may haveformula:

Still yet, the compound or its pharmaceutically acceptable salt may haveformula:

Further yet, the therapeutically effective amount of a compound or itspharmaceutically acceptable salt may be employed as an anti-cancer agentwherein cancer is selected from the group comprising lung cancer; breastcancer; colorectal cancer; anal cancer; pancreatic cancer; prostatecancer; ovarian carcinoma; liver and bile duct carcinoma; esophagealcarcinoma; non-Hodgkin's lymphoma; bladder carcinoma; carcinoma of theuterus; glioma, glioblastoma, medullablastoma, and other tumors of thebrain; myelofibrosis, kidney cancer; cancer of the head and neck; cancerof the stomach; multiple myeloma; testicular cancer; germ cell tumor;neuroendocrine tumor; cervical cancer; oral cancer, carcinoids of thegastrointestinal tract, breast, and other organs; signet ring cellcarcinoma; mesenchymal tumors including sarcomas, fibrosarcomas,haemangioma, angiomatosis, haemangiopericytoma, pseudoangiomatousstromal hyperplasia, myofibroblastoma, fibromatosis, inflammatorymyofibroblastic tumour, lipoma, angiolipoma, granular cell tumour,neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma,osteosarcoma, leiomyoma and/or a leiomysarcoma. Again still, thetherapeutically effective amount of the compound or its pharmaceuticallyacceptable salt may be administered to reduce cardiotoxicity sideeffects of chemotherapy and/or immunotherapy. Yet still again, thetherapeutically effective amount of the compound or its pharmaceuticallyacceptable salt may be administered as a single agent or in combinationwith chemotherapy, immunotherapy and/or radiotherapy in both adjuvantand neo-adjuvant settings. Again further, the therapeutically effectiveamount of the compound or its pharmaceutically acceptable salt may beadministered to induce metastasis inhibition. Furthermore, thetherapeutically effective amount of the compound or its pharmaceuticallyacceptable salt may be administered with a second therapeutic agentselected from an anti-cancer agent, an anti-inflammatory agent, ananti-hypertensive agent, an anti-fibrotic agent, an anti-angiogenicagent, and/or an immunosuppressive agent.

The current disclosure also provides a novel compound for inhibitinglysyl oxidases having formula:

Still further, the compound or its pharmaceutically acceptable salt maybe administered in a therapeutically effective amount to treat aneurodegenerative disease, an angiogenesis-related disease, Alzheimer'sdisease, fibrosis including: liver fibrosis, pulmonary fibrosis, renalfibrosis, myocardial fibrosis, cystic fibrosis, idiopathic pulmonaryfibrosis, radiation-induced fibrosis, ocular fibrosis, Peylony's diseaseand scleroderma, or respiratory disorders, abnormal wound healing andrepair, postoperative surgery, cardiac arrest-related fibrosis, excessor abnormal deposition of fibrotic material, all associated withdisorders such as Crohn's disease and inflammatory bowel disease, liver,kidney and pancreas fibrosis, diabetes, cerebral hemorrhage withamyloidosis, cardiac hypertrophy, Hutchinson-Gilford Progeria Syndrome,retinopathy, chemoresistance, and/or a kidney disorder including: kidneyfibrosis, renal fibrosis, acute kidney injury, chronic kidney disease,diabetic nephropathy, glomerulosclerosis, vesicoureteral reflux,tubulointerstitial renal fibrosis and/or glomerulonephritis. Yet again,the novel compound or its pharmaceutically acceptable salt may beadministered as a single agent or in combination with chemotherapy,immunotherapy and/or radiotherapy in both adjuvant and neo-adjuvantsettings. Still yet again, the compound or its pharmaceuticallyacceptable salt may be administered with a second therapeutic agentselected from an anti-cancer agent, an anti-inflammatory agent, ananti-hypertensive agent, an anti-fibrotic agent, an anti-angiogenicagent, and/or an immunosuppressive agent.

The current disclosure further provides a novel compound for inhibitinglysyl oxidases having formula:

Further, the novel compound or its pharmaceutically acceptable salt maybe administered in a therapeutically effective amount to treat aneurodegenerative disease, an angiogenesis-related disease, Alzheimer'sdisease, fibrosis including: liver fibrosis, pulmonary fibrosis, renalfibrosis, myocardial fibrosis, cystic fibrosis, idiopathic pulmonaryfibrosis, radiation-induced fibrosis, ocular fibrosis, Peylony's diseaseand scleroderma, or respiratory disorders, abnormal wound healing andrepair, postoperative surgery, cardiac arrest-related fibrosis, excessor abnormal deposition of fibrotic material, all associated withdisorders such as Crohn's disease and inflammatory bowel disease, liver,kidney and pancreas fibrosis, diabetes, cerebral hemorrhage withamyloidosis, cardiac hypertrophy, Hutchinson-Gilford Progeria Syndrome,retinopathy, chemoresistance, and/or a kidney disorder including: kidneyfibrosis, renal fibrosis, acute kidney injury, chronic kidney disease,diabetic nephropathy, glomerulosclerosis, vesicoureteral reflux,tubulointerstitial renal fibrosis and/or glomerulonephritis. Evenfurther, the compound or its pharmaceutically acceptable salt may beadministered as a single agent or in combination with chemotherapy,immunotherapy and/or radiotherapy in both adjuvant and neo-adjuvantsettings. Still yet again, the compound or its pharmaceuticallyacceptable salt may be administered with a second therapeutic agentselected from an anti-cancer agent, an anti-inflammatory agent, ananti-hypertensive agent, an anti-fibrotic agent, an anti-angiogenicagent, and/or an immunosuppressive agent.

These and other aspects, objects, features, and advantages of theexample embodiments will become apparent to those having ordinary skillin the art upon consideration of the following detailed description ofexample embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

An understanding of the features and advantages of the presentdisclosure will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the disclosure may be utilized, and the accompanyingdrawings of which:

FIG. 1 shows a diagram of extracellular matrix remodeling.

FIG. 2 shows graphical and visual analysis of tumor cell growth afterbirth and metastasis data resulting from treatment with CCT365623.

FIG. 3 shows an illustration of interaction between LOXL2, tumor cellsand collagen fibers.

FIG. 4 shows the molecular structure of PXS-5153A.

FIG. 5 shows one embodiment of a process for identification andcharacterization of novel LOX/LOXL inhibitors.

FIG. 6 shows relative lysyl oxidase activity and signal background forpotential inhibitors.

FIG. 7 shows a comparison of cell viability and lysyl oxidase activityfor target compounds.

FIG. 8 shows cell viability and lysyl oxidase activity for twentyspecific target compounds.

FIG. 9 shows a doxorubicin sensitization screen revealing five targetcompounds as doxorubicin sensitizers.

FIG. 10 shows LOX and LOX2 activity for compounds 6232 and 6229.

FIG. 11 shows SAR analysis of 6232 indicating further analog compounds.

FIG. 12 shows testing of analogs for lysyl oxidase activity and cellproliferation vis-à-vis drug dose.

FIG. 13 shows Table 1, Structure-Activity Relationship of4′-methyl-N2-phenyl-[4,5′-bithiazole]-2,2′-diamine Lox Inhibitors.

FIG. 14 shows testing of analogs 6403, 6405, and 6425 for toxicity innormal cells.

FIG. 15 shows in vivo toxicity testing for compound 6403.

FIG. 16 shows a graphical representation of growth inhibition forcompounds 6403 and 6415.

FIG. 17 shows reduced ECM crosslinking via use of compounds 6403 and5415.

FIG. 18 shows graphs of LOX and LOXL2 activity for compound 6415.

FIG. 19 shows graphical analysis of cell-based lysyl oxidase activityand cell viability assays in Caki-1 and ACHN cell lines treated withincreasing doses of BAPN and 6232.

FIG. 20 shows effects of ECM crosslinking in ACHN and Caki-1 cell lineswhen treated with compound 6232.

FIG. 21 shows decrease in ECM crosslinking upon treatment with compound6232.

FIG. 22 shows Expression of LOX family members in cancer vs normaltissue by using TCGA dataset.

FIG. 23 shows association of the expression of LOX, LOXL1 and LOXL2 withsurvival in breast, pancreatic and kidney cancer patients.

FIG. 24 shows LOX expression is increased in doxorubicin resistance andits high expression is associated with worse survival in chemotherapytreated TNBC patients.

FIG. 25 shows LOX inhibition overcomes doxorubicin resistance in 3Dculture.

FIG. 26 shows inhibiting LOX overcomes resistance in acquireddoxorubicin resistant MDA-MB-231 xenografts.

FIG. 27 shows targeting LOX in TNBC PDX organoids or tumors overcomesresistance.

FIG. 28 shows summary of our LOX or LOX family inhibitor discoverypipeline.

FIG. 29 shows characterization and selection of top hits from thehigh-throughput screen (HTS) of a diversified small-molecule library toidentify novel LOX inhibitors.

FIG. 30 shows LOX inhibitors generated based on SAR analysis.

FIG. 31 shows novel 6232 analogs, 6403 and 6415, effectively inhibitlysyl oxidase activity, overcomes doxorubicin resistance and reducecollagen cross-linking and fibronectin assembly.

FIG. 32 shows 6232 derivatives, 6403 and 6415, show no cytotoxicity innormal cells.

FIG. 33 shows while 6403 inhibits LOX recombinant protein, 6415 inhibitsboth LOX and LOXL2 recombinant proteins.

FIG. 34 shows 6232, 6403 and 6415 are more potent than LOX familyinhibitor, BAPN.

FIG. 35 shows 6403 and 6415 led to doxorubicin chemosensitization in 3Dculture of TNBC cell lines.

FIG. 36 shows combination of 6403 and 6415 with doxorubicin reduced thegrowth of the TNBC organoids.

FIG. 37 shows LOX inhibitors, 6232, 6403 and 6415, reduced the migrationcapacity of the TNBC cell lines.

FIG. 38 shows dose dependent inhibition of cellular LOX activity andgrowth inhibition in 3D upon treatment with 6232 and its analogs, 6403and 6415.in ACHN cancer cell line.

FIG. 39 shows inhibition of cellular LOX activity in pancreatic andkidney cancer cell lines.

FIG. 40 shows treatment with 6232, and its analogs 6403 and 6415inhibited the TGF-β-induced fibrosis in HFF cells.

FIG. 41 shows in vivo testing of 6403 with respect to PK and toxicity.

FIG. 42 shows efficacy of 6403 as a single agent in ccRC xenografts.

FIG. 43 shows that 6403 overcomes doxorubicin resistance without majortoxicity in a syngeneic mouse model, 4T1.

FIG. 44 shows Table 2, structure-activity relationship of4′-methyl-N2-phenyl-[4,5′-bithiazole]-2,2′-diamine Lox inhibitors.

FIG. 45 shows a synthesis scheme for Formula I.

FIG. 46 shows synthesis routes for analogs 6403 and 6415.

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Unless specifically stated, terms and phrases used in this document, andvariations thereof, unless otherwise expressly stated, should beconstrued as open ended as opposed to limiting. Likewise, a group ofitems linked with the conjunction “and” should not be read as requiringthat each and every one of those items be present in the grouping, butrather should be read as “and/or” unless expressly stated otherwise.Similarly, a group of items linked with the conjunction “or” should notbe read as requiring mutual exclusivity among that group, but rathershould also be read as “and/or” unless expressly stated otherwise.

Furthermore, although items, elements or components of the disclosuremay be described or claimed in the singular, the plural is contemplatedto be within the scope thereof unless limitation to the singular isexplicitly stated. The presence of broadening words and phrases such as“one or more,” “at least,” “but not limited to” or other like phrases insome instances shall not be read to mean that the narrower case isintended or required in instances where such broadening phrases may beabsent.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are cited todisclose and describe the methods and/or materials in connection withwhich the publications are cited. All such publications and patents areherein incorporated by references as if each individual publication orpatent were specifically and individually indicated to be incorporatedby reference. Such incorporation by reference is expressly limited tothe methods and/or materials described in the cited publications andpatents and does not extend to any lexicographical definitions from thecited publications and patents. Any lexicographical definition in thepublications and patents cited that is not also expressly repeated inthe instant application should not be treated as such and should not beread as defining any terms appearing in the accompanying claims Thecitation of any publication is for its disclosure prior to the filingdate and should not be construed as an admission that the presentdisclosure is not entitled to antedate such publication by virtue ofprior disclosure. Further, the dates of publication provided could bedifferent from the actual publication dates that may need to beindependently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Where a range is expressed, a further embodiment includes from the oneparticular value and/or to the other particular value. The recitation ofnumerical ranges by endpoints includes all numbers and fractionssubsumed within the respective ranges, as well as the recited endpoints.Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure. Forexample, where the stated range includes one or both of the limits,ranges excluding either or both of those included limits are alsoincluded in the disclosure, e.g., the phrase “x to y” includes the rangefrom ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’.The range can also be expressed as an upper limit, e.g., ‘about x, y, z,or less’ and should be interpreted to include the specific ranges of‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less thanx’, less than y′, and ‘less than z’. Likewise, the phrase ‘about x, y,z, or greater’ should be interpreted to include the specific ranges of‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greaterthan x’, greater than y′, and ‘greater than z’. In addition, the phrase“about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes“about ‘x’ to about ‘y’”.

It should be noted that ratios, concentrations, amounts, and othernumerical data can be expressed herein in a range format. It will befurther understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. Ranges can be expressed herein as from “about” one particularvalue, and/or to “about” another particular value. Similarly, whenvalues are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms a furtheraspect. For example, if the value “about 10” is disclosed, then “10” isalso disclosed.

It is to be understood that such a range format is used for convenienceand brevity, and thus, should be interpreted in a flexible manner toinclude not only the numerical values explicitly recited as the limitsof the range, but also to include all the individual numerical values orsub-ranges encompassed within that range as if each numerical value andsub-range is explicitly recited. To illustrate, a numerical range of“about 0.1% to 5%” should be interpreted to include not only theexplicitly recited values of about 0.1% to about 5%, but also includeindividual values (e.g., about 1%, about 2%, about 3%, and about 4%) andthe sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%;about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and otherpossible sub-ranges) within the indicated range.

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

As used herein, “about,” “approximately,” “substantially,” and the like,when used in connection with a measurable variable such as a parameter,an amount, a temporal duration, and the like, are meant to encompassvariations of and from the specified value including those withinexperimental error (which can be determined by e.g., given data set, artaccepted standard, and/or with e.g., a given confidence interval (e.g.,90%, 95%, or more confidence interval from the mean), such as variationsof +/−10% or less, +/−5% or less, +/−1% or less, and +/−0.1% or less ofand from the specified value, insofar such variations are appropriate toperform in the disclosure. As used herein, the terms “about,”“approximate,” “at or about,” and “substantially” can mean that theamount or value in question can be the exact value or a value thatprovides equivalent results or effects as recited in the claims ortaught herein. That is, it is understood that amounts, sizes,formulations, parameters, and other quantities and characteristics arenot and need not be exact, but may be approximate and/or larger orsmaller, as desired, reflecting tolerances, conversion factors, roundingoff, measurement error and the like, and other factors known to those ofskill in the art such that equivalent results or effects are obtained.In some circumstances, the value that provides equivalent results oreffects cannot be reasonably determined. In general, an amount, size,formulation, parameter or other quantity or characteristic is “about,”“approximate,” or “at or about” whether or not expressly stated to besuch. It is understood that where “about,” “approximate,” or “at orabout” is used before a quantitative value, the parameter also includesthe specific quantitative value itself, unless specifically statedotherwise.

As used herein, a “biological sample” may contain whole cells and/orlive cells and/or cell debris. The biological sample may contain (or bederived from) a “bodily fluid”. The present disclosure encompassesembodiments wherein the bodily fluid is selected from amniotic fluid,aqueous humour, vitreous humour, bile, blood serum, breast milk,cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph,perilymph, exudates, feces, female ejaculate, gastric acid, gastricjuice, lymph, mucus (including nasal drainage and phlegm), pericardialfluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skinoil), semen, sputum, synovial fluid, sweat, tears, urine, vaginalsecretion, vomit and mixtures of one or more thereof. Biological samplesinclude cell cultures, bodily fluids, and cell cultures from bodilyfluids. Bodily fluids may be obtained from a mammal organism, forexample by puncture, or other collecting or sampling procedures.

As used herein, “agent” refers to any substance, compound, molecule, andthe like, which can be administered to a subject on a subject to whichit is administered to. An agent can be inert. An agent can be an activeagent. An agent can be a primary active agent, or in other words, thecomponent(s) of a composition to which the whole or part of the effectof the composition is attributed. An agent can be a secondary agent, orin other words, the component(s) of a composition to which an additionalpart and/or other effect of the composition is attributed.

As used herein, “active agent” or “active ingredient” refers to asubstance, compound, or molecule, which is biologically active orotherwise that induces a biological or physiological effect on a subjectto which it is administered to. In other words, “active agent” or“active ingredient” refers to a component or components of a compositionto which the whole or part of the effect of the composition isattributed.

As used herein, “administering” refers to any suitable administrationfor the agent(s) being delivered and/or subject receiving said agent(s)and can be oral, topical, intravenous, subcutaneous, transcutaneous,transdermal, intramuscular, intra-joint, parenteral, intra-arteriole,intradermal, intraventricular, intraosseous, intraocular, intracranial,intraperitoneal, intralesional, intranasal, intracardiac,intraarticular, intracavernous, intrathecal, intravireal, intracerebral,and intracerebroventricular, intratympanic, intracochlear, rectal,vaginal, by inhalation, by catheters, stents or via an implantedreservoir or other device that administers, either actively or passively(e.g., by diffusion) a composition to the perivascular space andadventitia. For example, a medical device such as a stent can contain acomposition or formulation disposed on its surface, which can thendissolve or be otherwise distributed to the surrounding tissue andcells. The term “parenteral” can include subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, and intracranial injections orinfusion techniques. Administration routes can be, for instance,auricular (otic), buccal, conjunctival, cutaneous, dental,electro-osmosis, endocervical, endosinusial, endotracheal, enteral,epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration,interstitial, intra-abdominal, intra-amniotic, intra-arterial,intra-articular, intrabiliary, intrabronchial, intrabursal,intracardiac, intracartilaginous, intracaudal, intracavernous,intracavitary, intracerebral, intracisternal, intracorneal, intracoronal(dental), intracoronary, intracorporus cavernosum, intradermal,intradiscal, intraductal, intraduodenal, intradural, intraepidermal,intraesophageal, intragastric, intragingival, intraileal, intralesional,intraluminal, intralymphatic, intramedullary, intrameningeal,intramuscular, intraocular, intraovarian, intrapericardial,intraperitoneal, intrapleural, intraprostatic, intrapulmonary,intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular,intrathecal, intrathoracic, intratubular, intratumor, intratym panic,intrauterine, intravascular, intravenous, intravenous bolus, intravenousdrip, intraventricular, intravesical, intravitreal, iontophoresis,irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique,ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous,periarticular, peridural, perineural, periodontal, rectal, respiratory(inhalation), retrobulbar, soft tissue, sub arachnoid, subconjunctival,subcutaneous, sublingual, submucosal, topical, transdermal,transmucosal, transplacental, transtracheal, transtympanic, ureteral,urethral, and/or vaginal administration, and/or any combination of theabove administration routes, which typically depends on the disease tobe treated, subject being treated, and/or agent(s) being administered.

As used herein “cancer” can refer to one or more types of cancerincluding, but not limited to, acute lymphoblastic leukemia, acutemyeloid leukemia, adrenocortical carcinoma, Kaposi Sarcoma, AIDS-relatedlymphoma, primary central nervous system (CNS) lymphoma, anal cancer,appendix cancer, astrocytomas, atypical teratoid/Rhabdoid tumors, basalcell carcinoma of the skin, bile duct cancer, bladder cancer, bonecancer (including but not limited to Ewing Sarcoma, osteosarcomas, andmalignant fibrous histiocytoma), brain tumors, breast cancer, bronchialtumors, Burkitt lymphoma, carcinoid tumor, cardiac tumors, germ celltumors, embryonal tumors, cervical cancer, cholangiocarcinoma, chordoma,chronic lymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative neoplasms, colorectal cancer, craniopharyngioma,cutaneous T-Cell lymphoma, ductal carcinoma in situ, endometrial cancer,ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germcell tumor, extragonadal germ cell tumor, eye cancer (including, but notlimited to, intraocular melanoma and retinoblastoma), fallopian tubecancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoidtumor, gastrointestinal stromal tumors, central nervous system germ celltumors, extracranial germ cell tumors, extragonadal germ cell tumors,ovarian germ cell tumors, testicular cancer, gestational trophoblasticdisease, Hairy cell leukemia, head and neck cancers, hepatocellular(liver) cancer, Langerhans cell histiocytosis, Hodgkin lymphoma,hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrinetumors, kidney (renal cell) cancer, laryngeal cancer, leukemia, lipcancer, oral cancer, lung cancer (non-small cell and small cell),lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, metastaticsquamous cell neck cancer, midline tract carcinoma with and without NUTgene changes, multiple endocrine neoplasia syndromes, multiple myeloma,plasma cell neoplasms, mycosis fungoides, myelodyspastic syndromes,myelodysplastic/myeloproliferative neoplasms, chronic myelogenousleukemia, nasal cancer, sinus cancer, non-Hodgkin lymphoma, pancreaticcancer, paraganglioma, paranasal sinus cancer, parathyroid cancer,penile cancer, pharyngeal cancer, pheochromocytoma, pituitary cancer,peritoneal cancer, prostate cancer, rectal cancer, Rhabdomyosarcoma,salivary gland cancer, uterine sarcoma, Sezary syndrome, skin cancer,small intestine cancer, large intestine cancer (colon cancer), softtissue sarcoma, T-cell lymphoma, throat cancer, oropharyngeal cancer,nasopharyngeal cancer, hypoharyngeal cancer, thymoma, thymic carcinoma,thyroid cancer, transitional cell cancer of the renal pelvis and ureter,urethral cancer, uterine cancer, vaginal cancer, cervical cancer,vascular tumors and cancer, vulvar cancer, and Wilms Tumor.

As used herein, “chemotherapeutic agent” or “chemotherapeutic” refers toa therapeutic agent utilized to prevent or treat cancer.

As used herein, “control” can refer to an alternative subject or sampleused in an experiment for comparison purpose and included to minimize ordistinguish the effect of variables other than an independent variable.

The term “optional” or “optionally” means that the subsequent describedevent, circumstance or substituent may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances where it does not.

As used herein, “dose,” “unit dose,” or “dosage” can refer to physicallydiscrete units suitable for use in a subject, each unit containing apredetermined quantity of a pharmaceutical formulation thereofcalculated to produce the desired response or responses in associationwith its administration.

The term “molecular weight”, as used herein, can generally refer to themass or average mass of a material. If a polymer or oligomer, themolecular weight can refer to the relative average chain length orrelative chain mass of the bulk polymer. In practice, the molecularweight of polymers and oligomers can be estimated or characterized invarious ways including gel permeation chromatography (GPC) or capillaryviscometry. GPC molecular weights are reported as the weight-averagemolecular weight (M_(w)) as opposed to the number-average molecularweight (M_(n)). Capillary viscometry provides estimates of molecularweight as the inherent viscosity determined from a dilute polymersolution using a particular set of concentration, temperature, andsolvent conditions.

As used herein, “pharmaceutical formulation” refers to the combinationof an active agent, compound, or ingredient with a pharmaceuticallyacceptable carrier or excipient, making the composition suitable fordiagnostic, therapeutic, or preventive use in vitro, in vivo, or exvivo.

As used herein, “pharmaceutically acceptable carrier or excipient”refers to a carrier or excipient that is useful in preparing apharmaceutical formulation that is generally safe, non-toxic, and isneither biologically or otherwise undesirable, and includes a carrier orexcipient that is acceptable for veterinary use as well as humanpharmaceutical use. A “pharmaceutically acceptable carrier or excipient”as used in the specification and claims includes both one and more thanone such carrier or excipient.

As used herein, “polymer” refers to molecules made up of monomers repeatunits linked together. “Polymers” are understood to include, but are notlimited to, homopolymers, copolymers, such as for example, block, graft,random and alternating copolymers, terpolymers, etc. and blends andmodifications thereof. “A polymer” can be a three-dimensional network(e.g., the repeat units are linked together left and right, front andback, up and down), a two-dimensional network (e.g., the repeat unitsare linked together left, right, up, and down in a sheet form), or aone-dimensional network (e.g., the repeat units are linked left andright to form a chain). “Polymers” can be composed, natural monomers orsynthetic monomers and combinations thereof. The polymers can bebiologic (e.g., the monomers are biologically important (e.g., an aminoacid), natural, or synthetic.

As used herein, the term “radiation sensitizer” refers to agents thatcan selectively enhance the cell killing from irradiation in a desiredcell population, such as tumor cells, while exhibiting no single agenttoxicity on tumor or normal cells.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed by the term “subject”.

As used herein, “substantially pure” can mean an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises about 50 percent of all species present. Generally, asubstantially pure composition will comprise more than about 80 percentof all species present in the composition, more preferably more thanabout 85%, 90%, 95%, and 99%. Most preferably, the object species ispurified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single species.

As used interchangeably herein, the terms “sufficient” and “effective,”can refer to an amount (e.g., mass, volume, dosage, concentration,and/or time period) needed to achieve one or more desired and/or statedresult(s). For example, a therapeutically effective amount refers to anamount needed to achieve one or more therapeutic effects.

As used herein, “tangible medium of expression” refers to a medium thatis physically tangible or accessible and is not a mere abstract thoughtor an unrecorded spoken word. “Tangible medium of expression” includes,but is not limited to, words on a cellulosic or plastic material, ordata stored in a suitable computer readable memory form. The data can bestored on a unit device, such as a flash memory or CD-ROM or on a serverthat can be accessed by a user via, e.g., a web interface.

As used herein, “therapeutic” can refer to treating, healing, and/orameliorating a disease, disorder, condition, or side effect, or todecreasing in the rate of advancement of a disease, disorder, condition,or side effect. A “therapeutically effective amount” can therefore referto an amount of a compound that can yield a therapeutic effect.

As used herein, the terms “treating” and “treatment” can refer generallyto obtaining a desired pharmacological and/or physiological effect. Theeffect can be, but does not necessarily have to be, prophylactic interms of preventing or partially preventing a disease, symptom orcondition thereof, such as cancer and/or indirect radiation damage. Theeffect can be therapeutic in terms of a partial or complete cure of adisease, condition, symptom or adverse effect attributed to the disease,disorder, or condition. The term “treatment” as used herein covers anytreatment of cancer and/or indirect radiation damage, in a subject,particularly a human and/or companion animal, and can include any one ormore of the following: (a) preventing the disease or damage fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it; (b) inhibiting the disease, i.e.,arresting its development; and (c) relieving the disease, i.e.,mitigating or ameliorating the disease and/or its symptoms orconditions. The term “treatment” as used herein can refer to boththerapeutic treatment alone, prophylactic treatment alone, or boththerapeutic and prophylactic treatment. Those in need of treatment(subjects in need thereof) can include those already with the disorderand/or those in which the disorder is to be prevented. As used herein,the term “treating”, can include inhibiting the disease, disorder orcondition, e.g., impeding its progress; and relieving the disease,disorder, or condition, e.g., causing regression of the disease,disorder and/or condition. Treating the disease, disorder, or conditioncan include ameliorating at least one symptom of the particular disease,disorder, or condition, even if the underlying pathophysiology is notaffected, such as treating the pain of a subject by administration of ananalgesic agent even though such agent does not treat the cause of thepain.

As used herein, the terms “weight percent,” “wt %,” and “wt. %,” whichcan be used interchangeably, indicate the percent by weight of a givencomponent based on the total weight of a composition of which it is acomponent, unless otherwise specified. That is, unless otherwisespecified, all wt % values are based on the total weight of thecomposition. It should be understood that the sum of wt % values for allcomponents in a disclosed composition or formulation are equal to 100.Alternatively, if the wt % value is based on the total weight of asubset of components in a composition, it should be understood that thesum of wt % values the specified components in the disclosed compositionor formulation are equal to 100.

As used herein, “water-soluble”, generally means at least about 10 g ofa substance is soluble in 1 L of water, i.e., at neutral pH, at 25° C.

Various embodiments are described hereinafter. It should be noted thatthe specific embodiments are not intended as an exhaustive descriptionor as a limitation to the broader aspects discussed herein. One aspectdescribed in conjunction with a particular embodiment is not necessarilylimited to that embodiment and can be practiced with any otherembodiment(s). Reference throughout this specification to “oneembodiment”, “an embodiment,” “an example embodiment,” means that aparticular feature, structure or characteristic described in connectionwith the embodiment is included in at least one embodiment of thepresent disclosure. Thus, appearances of the phrases “in oneembodiment,” “in an embodiment,” or “an example embodiment” in variousplaces throughout this specification are not necessarily all referringto the same embodiment, but may. Furthermore, the particular features,structures or characteristics may be combined in any suitable manner, aswould be apparent to a person skilled in the art from this disclosure,in one or more embodiments. Furthermore, while some embodimentsdescribed herein include some but not other features included in otherembodiments, combinations of features of different embodiments are meantto be within the scope of the disclosure. For example, in the appendedclaims, any of the claimed embodiments can be used in any combination.

All patents, patent applications, published applications, andpublications, databases, websites and other published materials citedherein are hereby incorporated by reference to the same extent as thougheach individual publication, published patent document, or patentapplication was specifically and individually indicated as beingincorporated by reference.

Kits

Any of the compounds and/or formulations described herein can bepresented as a combination kit. As used herein, the terms “combinationkit” or “kit of parts” refers to the compounds, compositions,formulations, particles, cells and any additional components that areused to package, sell, market, deliver, and/or administer thecombination of elements or a single element, such as the activeingredient, contained therein. Such additional components include, butare not limited to, packaging, syringes, blister packages, bottles, andthe like. When one or more of the compounds, compositions, formulations,particles, cells, described herein or a combination thereof (e.g.,agent(s)) contained in the kit are administered simultaneously, thecombination kit can contain the active agent(s) in a single formulation,such as a pharmaceutical formulation, (e.g., a tablet, liquidpreparation, dehydrated preparation, etc.) or in separate formulations.When the compounds, compositions, formulations, particles, and cellsdescribed herein or a combination thereof and/or kit components are notadministered simultaneously, the combination kit can contain each agentor other component in separate pharmaceutical formulations. The separatekit components can be contained in a single package or in separatepackages within the kit.

In some embodiments, the combination kit also includes instructionsprinted on or otherwise contained in a tangible medium of expression.The instructions can provide information regarding the content of thecompounds and/or formulations, safety information regarding the contentof the compounds and formulations (e.g., pharmaceutical formulations),information regarding the dosages, indications for use, and/orrecommended treatment regimen(s) for the compound(s) and/orpharmaceutical formulations contained therein. In some embodiments, theinstructions can provide directions and protocols for administering thecompounds and/or formulations described herein to a subject in needthereof. In some embodiments, the instructions can provide one or moreembodiments of the methods for administration of a pharmaceuticalformulation thereof such as any of the methods described in greaterdetail elsewhere herein.

While dramatic advances have been made in the treatment and curation ofcancer, disease progression following first-line treatment, accompaniedby the spread of cancer to distant organs is inevitable in many casesand associated with cancer mortality. Proteins that re-model theextracellular matrix (ECM) which surrounds the tumors and helps themgrow and invade distant organs are attractive therapeutic targets notonly for cancer but also for other diseases involving excessive ECMdeposition such as fibrotic diseases, neurodegenerative andcardiovascular diseases.

Lysyl oxidase family proteins are composed of five members (LOX and fourrelated enzymes, LOXL1-4). The main function of lysyl oxidases is tocatalyze the crosslinking of extracellular matrix (ECM) components,mainly collagens and elastin, and thus re-modelling the ECM. Given thecrucial roles they play in maintaining tissue homeostasis, deregulationof lysyl oxidases may lead to various diseases, including cancer, organfibrosis, neurodegenerative and cardiovascular diseases. Each LOX enzymehas several overlapping as well as unique functions. LOX and LOXL2proteins have been implicated in progression and metastasis of severaltypes of cancers, such as breast, pancreas, and lung adenocarcinomas.Their role in promoting breast cancer metastasis has been associatedwith their ability to increase the stiffness of the tumors and toenhance collagen crosslinking at the metastatic site, thus preparing therequired niche for metastatic out-growth. They can also stimulateendothelial cells and promote angiogenesis. In addition, published datafrom us and others have implicated a role of LOX proteins in therapyresistance of cancers. LOX/LOXLs can regulate cancer cell invasion bypromoting epithelial-mesenchymal transition (EMT), stabilization of EMTtranscription factors or repression of epithelial gene transcription.They also have non-canonical functions such as regulation of genetranscription. Most importantly, high expression of LOX/LOXLs isassociated with poor prognosis in different cancers. In addition tocancer, LOX family proteins have also been linked to fibrotic disease,extending the applicability of LOX/LOXL targeting strategies. In aneffort to identify novel compounds targeting LOX proteins, the inventorsexecuted a high-throughput screening program and identified a number ofinitial hit compounds which reduced lysyl oxidase activity in a cellularassay with minimum cell toxicity. A fresh batch of the shortlistedcompounds were then purchased, and the dose-dependent effects on cellviability and lysyl oxidase activity were validated. The inventors thentested the effects of compounds on chemotherapy sensitization and alsotested the direct inhibitory effects on LOX and LOXL2 activity usingrecombinant proteins. A final filter was the assessment of the compoundsdrug-likeness and the ease of making new analogues through syntheticchemistry. One series based upon a4′-methyl-N2-phenyl-[4,5′-bithiazole]-2,2′-diamine core structurefulfilled all of these criteria and our current lead inhibitor from thisseries is4-((2′-amino-4′-methyl-[4,5′-bithiazol]-2-yl)(methyl)amino)phenol. Thiscompound is a non-selective inhibitor of the LOX family of enzymes.

In cancer, ECM is frequently remodeled by cancer cells themselves ortumor-associated stromal cells, leading to increased tumor stiffness andthus, enhanced proliferation, migration and invasion. ECM remodeling atthe metastatic site can also trigger colonization and metastaticoutgrowth. Besides cancer, disruption of ECM homeostasis may also leadto organ fibrosis, neurodegenerative and cardiovascular diseases. FIG. 1shows a diagram of extracellular matrix remodeling. See, Hung-Yu Lin etal, 2020, International Journal of Molecular Sciences.

Given the highly critical functions they have in ECM remodeling,targeting LOX/LOXLs is an attractive therapeutic strategy that willbenefit the treatment of ECM-driven diseases. BAPN is the most widelyused LOX family inhibitor. However, due to its simple chemicalstructure, it is highly unspecific leading to severe toxicity. It alsolacks sites amenable for chemical modification, and thus precludespreclinical optimization. A few studies reported LOXL2-specificinhibitor and LOX/LOXL inhibitors. See Tang et al., Lysyl oxidase drivestumor progression by trapping EGF receptors at the cell surface (2017).Tang et al. developed an orally bioavailable LOX/LOXL2 inhibitor namedCCT365623. This inhibitor is an aminomethylenethiophene (AMT) basedinhibitor. Treatment with CCT365623, daily by oral gavage with 70 mg/kgdose decreased tumor growth and reduced metastasis in breast cancermodels. See FIG. 2 , which shows graphical and visual analysis of tumorcell growth after birth and metastasis data resulting from treatmentwith CCT365623. See, Tang et al., Lysyl Oxidase Drives Tumor Progressionby Trapping EFT Receptors at the Cell Surface, DOI: 10.1038/ncomms14909.

Further, the non-competitive LOXL2 targeting humanized monoclonalantibody, Simtuzumab showed beneficial effects in various preclinicalmodels of fibrosis and cancer. It has also been tested in severalclinical trials in the context of fibrotic diseases as well as cancer.For cancer, a phase II clinical trial was conducted with Simtuzumab incombination with gemcitabine to treat pancreatic cancer patients(NCT01472198). Although the antibody was well-tolerated, the clinicalbenefit was minimal due to lack of efficacy. See FIG. 3 , which shows anillustration of interaction between LOXL2, tumor cells and collagenfibers. See, Sandra Ferreira S. et al, Antioxidants, 2021. The lack ofefficacy with LOXL2-targeting antibody may be attributed to the factthat Simtuzumab only targets extracellular LOXL2 which is apparently noteffective enough owing to several intracellular functions of the LOXfamily proteins (Sandra Ferreira S. et al, Antioxidants, 2021).

Therefore, targeting LOX enzymes with small molecule inhibitors thatwill inhibit both the intracellular and extracellular LOX functionsshould be much more effective. FIG. 4 shows the molecular structure ofPXS-5153A. See, (Schilter H et al., 2018, J. Cell. Mol. Med). The smallmolecule PXS-5153A demonstrated complete and irreversible enzymeinhibition for LOXL2 and LOXL3. PXS-5153A was shown to reduceLOXL2-mediated collagen oxidation and cross-linking, in a dose-dependentmanner in vitro. This dual LOXL2/LOXL3 inhibitor has shown beneficialeffects in models of liver fibrosis and myocardial infarction (SchilterH et al, 2018, J. Cell. Mol. Med.). Pharmaxis has developed an orallybioavailable pan-lysyl oxidase inhibitor PXS-5505, inhibiting all lysyloxidase family members. The compound has shown significant reductions infibrosis in in vivo models of kidney fibrosis, lung fibrosis,myelofibrosis (a rare type of blood cancer) and pancreatic cancer. Italso demonstrated an excellent safety profile and was well tolerated inhealthy male volunteers (How, J. et al, 2020, Blood). A Phase 1c/2aclinical trial in myelofibrosis patients is now planned whose resultsare expected by 2023.

The current disclosure, inter alia, provides methods for identificationand characterization of novel LOX/LOXL inhibitors FIG. 5 shows oneembodiment of a process for identification and characterization of novelLOX/LOXL inhibitors. Twenty compounds from a library of drug-likemolecules and bioactive compounds were identified as potential lysyloxidase inhibitors that cause no major cellular cytotoxicity. 4,480small molecules from EXPRESS-Pick diversified library by ChemBridge anda Tocriscreen library of bioactive compounds by Tocris (1,280compounds). FIG. 6 shows relative lysyl oxidase activity and signalbackground for potential inhibitors. FIG. 7 shows a comparison of cellviability and lysyl oxidase activity for target compounds. FIG. 8 showscell viability and lysyl oxidase activity for twenty specific targetcompounds. Further, nineteen short-listed compounds were freshlyobtained, and their effects on cell viability and lysyl oxidase activitywere validated. A doxorubicin sensitization screen was performed withthe shortlisted nineteen (19) candidates, and five (5) compounds wereidentified as doxorubicin sensitizers. FIG. 9 shows a doxorubicinsensitization screen revealing five target compounds as doxorubicinsensitizers. Among five (5) candidates, two (2) compounds (6232 and6229) decreased the activity of both the recombinant LOX and LOXL2proteins, although LOXL2 inhibition was weaker as compared to BAPN. FIG.10 shows LOX and LOX2 activity for compounds 6232 and 6229. SAR analyseswith 6232 led to several analogs: lysyl oxidase activity inhibition incell-based assay. FIG. 11 shows SAR analysis of 6232 indicating furtheranalog compounds. Six (6) of the promising analogs were further testedat multiple doses. FIG. 12 shows testing of analogs for lysyl oxidaseactivity and cell proliferation vis-à-vis drug dose. FIG. 13 shows Table1, Structure-Activity Relationship of4′-methyl-N2-phenyl-[4,5′-bithiazole]-2,2′-diamine Lox Inhibitors.Shortlisted analogs (6403, 6405 and 6425) were tested in terms oftoxicity in normal cells. FIG. 14 shows testing of analogs 6403, 6405,and 6425 for toxicity in normal cells. No significant effect on theviability of three different normal cell lines. FIG. 15 shows in vivotoxicity testing for compound 6403. FIG. 16 shows a graphicalrepresentation of growth inhibition for compounds 6403 and 6415. Nomajor change in body weight or blood cell count were observed even athigher doses. FIG. 17 shows reduced ECM crosslinking via use ofcompounds 6403 and 5415. FIG. 18 shows graphs of LOX and LOXL2 activityfor compound 6415.

The inhibitory effect of our parental compound, 6232 has also beentested in ccRCCs having pseudo-hypoxia (i.e., constitutive activation ofHIF1α and HIF2α independent of the oxygen level). Cell-based lysyloxidase activity and cell viability assays in Caki-1 and ACHN cell linestreated with increasing doses of BAPN and 6232. FIG. 19 shows graphicalanalysis of cell-based lysyl oxidase activity and cell viability assaysin Caki-1 and ACHN cell lines treated with increasing doses of BAPN and6232. FIG. 20 shows effects of ECM crosslinking in ACHN and Caki-1 celllines when treated with compound 6232. FIG. 21 shows decrease in ECMcrosslinking upon treatment with compound 6232.

As the available LOX inhibitors are either not very potent or havetoxicity issues, the inventors developed a robust screening pipeline toidentify potent and safe LOX-specific and pan-LOX inhibitors. Ahigh-throughput cell-based LOX activity (Abcam) screen was done for morethan 5,000 drug-like compounds. The toxic molecules were counterscreened with cell cytotoxicity assay and filtered out. Among the mostpotent hits (20 in total), 5 of them were shortlisted based on theirability to sensitize TNBC cells to doxorubicin, to cross-link collagen-Iand tested in recombinant LOX and LOXL2 protein-based activity assays todetermine their on-target activity and selectivity. One of the compounds(6232 (phenylbisthiazole diamine core structure)) was selected for SARand lead optimization studies based on favorable chemistry towardsdrug-likeness, synthetic feasibility and potential for creating noveland chemically distinct LOX inhibitors. After the SAR analysis, severalanalogs showed similar or increased activity relative to 6232 and both6403 and 6415, derivatives of 6232, led to doxorubicin sensitization.Lysyl oxidase activity assay with recombinant proteins revealed that6403, a derivative of 6232 (NO2 replaced with acetamide), is a morepotent and selective inhibitor against LOX compared to LOXL2, while 6415is more like a LOX/LOXL2 dual inhibitor. Importantly, 6403 showed morepotent cellular lysyl oxidase inhibition (IC50=1.3 uM for LOXinhibition) when compared to BAPN (IC50>100 uM). 6403, showed favorablepharmacokinetic (PK) properties in mice and did not lead to anyobservable organ damage. As a single agent, the inventors tested our LOXinhibitors on kidney cancer in vivo xenograft models. 6403 successfullyreduced the tumor growth of LOX-expressing ACHN xenografts withoutimpacting body weight. Finally, the inventors tested the chemosensitizereffect of 6403 in a syngeneic TNBC murine tumor model, 4T1 which has anintact immune system. Combination of 6403 with doxorubicin significantlyreduced tumor growth in this LOX-expressing model compared to 6403 anddoxorubicin alone groups with no significant change in body weights andblood counts after 21-day treatment. Overall, the inventors obtainedboth LOX-specific and broader LOX family inhibitors that are novel andhave considerable potential for drug development.

Among the LOX family members, expression of LOX, LOXL1 and LOXL2 washigher in broad spectrum of cancers compared to normal tissue in TheCancer Genome Atlas (TCGA) dataset, see FIG. 22 at A. LOX, LOXL1 andLOXL2 are highly expressed in TNBCs vs normal breast, see FIG. 22 at B,pancreatic tumors vs. normal tissue and kidney cancer vs. normal tissue,see FIG. 22 at C. These are the tumors known to be stroma-rich andfibrotic.

In the same TCGA dataset, higher expression of LOX, LOXL1 and LOXL2associates with worse disease-free survival (DFS) in kidney renal cellcarcinoma (KIRC). Furthermore, higher expression of LOX and LOXL2associates with worse relapse-free survival (RFS) in pancreaticadenocarcinoma (PAAD) (in the TCGA dataset) and in chemotherapy-treatedbreast cancer (KM plotter database), see FIG. 23 at A, B and C. Theseresults suggest that higher expression of LOX family members, especiallyLOX and LOXL2, is associated with worse disease progression in cancers,especially with stiff tumors.

The current inventors identified the hypoxia inducible factor alpha(HIF1α)-induced LOX as being significantly overexpressed, along withincreased fibrillar collagen in tumors of our clinically relevant invivo model of doxorubicin resistance, see FIG. 24 at A and B. theinventors demonstrated that higher LOX mRNA/protein levels aresignificantly associated with worse disease-free survival (DFS) inchemotherapy treated TNBC patients using multiple published datasets andour own patient cohort, see FIG. 24 at C and D.

The non-specific LOX family inhibitor, BAPN re-sensitized threedifferent collagen I (LOX substrate)-embedded human TNBC cell lines(MDA-MB-231, FIG. 25 at A, MDA-MB-15714 and MDA-MB-43614) todoxorubicin. These results were also recapitulated by specific LOXinhibition upon siRNA-methated knockdown, See FIG. 25 at B.Immunofluorescence (IF) staining of collagen and fibronectin uponincubation of BAPN-treated MDA-MB-231 cells with rat tail collagen I orECM produced by human foreskin fibroblast (HFF) cells demonstratedreduced ECM deposition and assembly, leading to enhanced drugpenetration upon LOX inhibition, See FIG. 25 at C and D. This led tode-activation of downstream FAK/Src signaling, see FIG. 25 at E, andincreased apoptosis, see FIG. 25 at F. Importantly, inhibiting FAK orSrc kinases recapitulated LOX inhibition-mediated chemosensitization,see FIG. 25 at G.

To test the ability of LOX inhibition to overcome doxorubicin resistancein chemotherapy-refractory TNBC in vivo, the inventors first treatedMDA-MB-231 xenografts with doxorubicin until an accelerated tumor growthwas achieved. At this point, BAPN (100 mg/kg) was added to half of thedoxorubicin-resistant tumors while the rest continued to receivedoxorubicin alone (2.5 mg/kg). Strikingly, the addition of LOX inhibitorled to a significant decrease in tumor growth, see FIG. 26 at A, andimproved survival. the inventors confirmed the reduction in LOXactivity, see FIG. 26 at B, and fibrillar collagen, see FIG. 26 at C.

To assess the clinical relevance of targeting LOX, the inventorsobtained RNA-Seq data from 15 patient-derived xenograft (PDX) models ofTNBC from Jackson Lab along with their drug response data, see FIG. 27at A, and performed a gene expression-based assessment of hypoxia andfocal adhesion (FA) scores for each model and correlated these scoreswith LOX expression, see FIG. 27 at B. Importantly, expression of LOXhad a good correlation (r=0.649 and r=0.625) with both scores. Based onthe RNA-Seq analysis and drug response data, the inventors selected oneresistant model, TM01278 that is resistant to doxorubicin and expresseshigh levels of LOX with high hypoxia and focal adhesion scores,Combination of LOX inhibitor with doxorubicin significantly decreasedtumor organoid size in 3D organoid culture with collagen I, see FIG. 27at C, and reduced tumor growth, see FIG. 27 at D. Importantly, LOXactivity and fibrillar collagen content were reduced upon LOX inhibitionwith BAPN, see FIG. 27 at E, which was accompanied by enhanceddoxorubicin penetration, see FIG. 27 at F, and reduced FAK/Srcsignaling, see FIG. 27 at G.

As the available LOX inhibitors are either not very potent or havetoxicity, the inventors developed a robust screening pipeline shown inFIG. 28 to identify potent and safe LOX-specific and pan-LOX inhibitors.Briefly, a high-throughput cell-based LOX activity (Abcam) screen (HTS)was done for more than 5,000 drug-like compounds from the EXPRESS-Pickdiversified library (4,480 molecules, ChemBridge) and Tocriscreenlibrary of bioactive compounds (1,280 compounds, Tocris), each at asingle concentration of 10 μM. The screen identified 25 compounds fromChemBridge library and 56 hits from Tocris library that successfullyinhibited >90% of LOX activity. The toxic molecules were counterscreened with cell cytotoxicity assay and filtered out.

The most potent hits (20 in total) were then verified by re-orderingfresh compounds and testing them in the LOX activity and cell viabilityassays, see FIG. 29 at A. Among these 20 compounds, 5 of them wereshortlisted based on their ability to sensitize TNBC cells todoxorubicin, see FIG. 29 at B. These 5 compounds were further tested inrecombinant LOX and LOXL2 protein-based activity assays to determinetheir on-target activity and selectivity. Two compounds (6229 and 6232)were shown to strongly inhibit recombinant LOX, and moderately inhibitLOXL2 at 10 μM, a concentration that is 1000× lower than required forLOX family inhibitor, BAPN, see FIG. 29 at C and D.

A diverse set of analogs of 6232 (phenylbisthiazole diamine corestructure, see FIG. 30 ) were commercially available from ChemBridge andin total 19 compounds were sourced. The inventors found that severalanalogs possessed similar or increased activity relative to 6232 andprovided initial insights into the SAR (see Table 2—FIG. 44 , FIG. 30and FIG. 31 at A). These data pointed to the importance of the freeamino group on the terminal thiazole ring and suggested that a varietyof substituents could be tolerated on the aniline ring without loss ofactivity. Of the 19 analogs tested, 6403 and 6415 were the most potentwith cellular LOX activity of 1.3 μM and 3.5 μM respectively andtherefore these analogs were considered preliminary lead compounds. Theinventors also synthesized two new derivatives based on thephenylbisthiazole diamine core structure. Compounds 6425 and 6426included additional variations of the R₄ group (6425 ismethoxypropanamide, 6426 is pentamide) and showed high potency withcellular LOX activity of 7.5 μM and 2 μM, respectively. Both 6425 and6426 are novel compounds and have not been reported in chemicalabstracts.

Treatment with both 6403 and 6415 led to doxorubicin sensitization, FIG.31 at B, and reduced collagen crosslinking/fibronectin assembly, seeFIG. 31 at C. Furthermore, cytotoxicity of 6403 and 6415 was tested onnormal cell lines; MCF12A, HUVEC and HFF, see FIG. 32 , and nosignificant change in cell viability was detected.

Lysyl oxidase activity assay with recombinant proteins revealed that6403 is a more potent and selective inhibitor against LOX compared toLOXL2, meeting our requirement for LOX specificity, see FIG. 33 at A,while 6415 is more like a LOX/LOXL2 inhibitor, see FIG. 33 at B.

6232, 6403 and 6415 showed more potent cellular lysyl oxidase inhibition(e.g., IC50=1.3 uM for 6403) when compared to BAPN (IC50>100 uM), seeFIG. 34 .

Treatment with both 6403 and 6415 led to doxorubicin sensitization incollagen I-embedded MDA-MB-231 and HCC1143 TNBC cell lines, see FIG. 35.

Then, the inventors generated organoid cultures of thisdoxorubicin-resistant PDX tumors (TM01278) and demonstrated thatcombination of LOX inhibitors, 6403 and 6415, with doxorubicinsignificantly decreased organoid size, see FIG. 36 at A, and organoidviability, see FIG. 36 at B, compared to single agent treatments after 9days of treatment.

The effects of LOX inhibitors, 6232, 6403 and 6415, were tested on cellmigration by using RTCA-CIM plate (Roche, USA). All three compoundsinhibited the migration capacity of MDA-MB-231 cells, 6403 being themost effective one in this cell line, see FIG. 37 .

Previous studies showed that LOX is overexpressed in different cancersand strongly associated with poor outcome in patients with pancreatic,see Miller, B. W. et al. Targeting the LOX/hypoxia axis reverses many ofthe features that make pancreatic cancer deadly: inhibition of LOXabrogates metastasis and enhances drug efficacy. EMBO Mol Med 7,1063-1076, doi:10.15252/emmm.201404827 (2015). and kidney, see Li, T. etal. Lysyl oxidase family members in urological tumorigenesis andfibrosis. Oncotarget 9, 20156-20164, doi:10.18632/oncotarget.24948(2018), tumors, making it an attractive therapeutic target for theseclinically challenging diseases. The inventors observed dose dependentcellular lysyl oxidase activity inhibition, see FIG. 38 at A, anddecrease in cell viability in 3D culture, see FIG. 38 at B, upontreatment with 6232, 6403 and 6415 in ACHN kidney cancer cell line, seeFIG. 38 . In addition to ACHN, treatment with 6232, 6403 and 6415inhibited cellular LOX activity of another kidney cancer cell lineCaki-1 and pancreatic cancer cell lines, Mia-Paca-2 and PANC1, see FIG.39 . Inhibition of LOX activity via our new lead inhibitors will notonly be beneficial for TNBC, but also other tumor types characterized byhigh stiffness and chemoresistance, such as pancreatic cancer, orLOX-driven cancers, such as kidney cancer in future.

The inventors tested the effects of our LOX inhibitors on the TGF-betainduced fibrosis. Immunofluorescence staining of fibrosis markers aresubstantially reduced upon treatment with LOX inhibitors, see FIG. 40 .ALK5 inhibitor was used as a positive control.

Our lead LOX inhibitor 6403 showed favorable pharmacokinetic (PK)properties in mice, with a high Area Under the Curve (AUC), longhalf-life (T½), high maximum concentration (Cmax), and volume ofdistribution (Vd) while having low clearance (Cl), see FIG. 41 at A, anddid not lead to any observable organ damage, see FIG. 41 at B or bodyweight change, see FIG. 41 at C. Importantly, our parental molecule6232, and its derivatives 6403 and 6415, did not inhibit monoamineoxidase A or B (MAO-A/-B) activity, see FIG. 41 at D, in cell-freeenzymatic activity assay, demonstrating its selectivity towards LOXenzymes.

6403 successfully reduced the tumor growth of LOX-expressing ACHN ccRCCxenografts without impacting body weight and counts of major bloodcells, see FIG. 42 .

Finally, the inventors tested the chemosensitizing effect of 6403 in asyngeneic TNBC tumor model, 4T1 which has an intact immune system.Combination of 6403 with doxorubicin significantly reduced tumor growthin this LOX-expressing model compared to 6403 and doxorubicin alonegroups, see FIG. 43 at A and B, with no significant change in bodyweights and blood counts after 21-day treatment, see FIG. 43 at C and D.

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are effective asinhibitors of LOX or LOX family activity. Such compounds have thegeneral formula I:

or a pharmaceutically acceptable salt thereof, wherein each variable isas defined and described herein. Compounds of the present invention, andpharmaceutically acceptable compositions thereof, are useful fortreating a variety of diseases, disorders, or conditions, associatedwith LOX activity. Such diseases, disorders, or conditions include thosedescribed herein. Compounds provided by this invention are also usefulfor the study of LOX enzymes in biological and pathological phenomena.

I or a pharmaceutically acceptable salt thereof, wherein:

Q1 is ═N— or ═CH—

Q2 is NH, O or S

Q3 is ═N— or ═CH—

Q4 is NH, O or S and Q5 is ═N— or ═CH—

Q4=N— or ═CH— and Q5 is NH, O or S

Q6 is ═N— or ═CH—R4

Q7 is ═N— or ═CH—R5

Q8 is ═N— or ═CH—R6

Q9 is ═N— or ═CH—R7

R1-R7 is independently halogen, —CN, —NO₂, —NH₂, —NHR, —CH₂R, —OR, —SR,—NR₂, —S(O)₂R, —S(O)₂NR₂, —S(O)R, —C(O)R, —C(O)OR, —C(O)NR₂,—C(O)N(R)OR, —N(R)C(O)OR, —N(R)C(O)NR₂, or —N(R)S(O)₂R;

Where each R is independently hydrogen, or an optionally substitutedgroup selected from C1-6 aliphatic, phenyl, 4-7 membered saturated orpartially unsaturated heterocyclic having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, or 5-6 membered heteroarylring having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or: two R groups on the same nitrogen are takentogether with their intervening atoms to form a 4-7 membered saturated,partially unsaturated, or heteroaryl ring having 0-3 heteroatoms, inaddition to the nitrogen, independently selected from nitrogen, oxygen,or sulfur, see Table 2, FIG. 44 .

The chemical structure for 6232 is:

The chemical structure for 6403 is:

The chemical structure for 6405 is:

The chemical structure for 6415 is:

FIG. 45 shows a synthesis scheme for Formula I. FIG. 46 shows synthesisroutes for analogs 6403 and 6415.

Figure Legends

FIG. 1 shows a diagram of extracellular matrix remodeling (Hung-Yu Linet al, 2020, International Journal of Molecular Sciences).

FIG. 2 shows graphical and visual analysis of tumor cell growth afterbirth and metastasis data resulting from treatment with CCT365623 (Tang,HaoRan, et al., 2017, Nature Communications). Treatment with CCT365623,daily by oral gavage with 70 mg/kg dose decreased tumor growth andreduced metastasis in breast cancer models.

FIG. 3 shows an illustration of interaction between LOXL2, tumor cellsand collagen fibers (Ferreira, Sandra, et al., 2021, Antioxidants).

FIG. 4 shows the molecular structure of PXS-5153A (Schiller H et al,2018, J. Cell. Mol. Med).

FIG. 5 shows one embodiment of a process for identification andcharacterization of novel LOX/LOXL inhibitors.

FIG. 6 shows the preliminary testing of LOX activity assay for compoundscreen. Left panel shows the relative lysyl oxidase activity and rightpanel shows signal-to-background ratio for the general LOX inhibitor,BAPN.

FIG. 7 shows a comparison of percentage cell viability and lysyl oxidaseactivity for target compounds using MDA-MB-231 cells. 20 compounds froma library of drug-like molecules and bioactive compounds were identifiedas potential lysyl oxidase inhibitors that cause no major cellularcytotoxicity.

FIG. 8 shows the validation of the effects of 19 short-listed compoundsthat were freshly obtained on cell viability and lysyl oxidase activity.

FIG. 9 shows a doxorubicin sensitization screen revealing five targetcompounds as doxorubicin sensitizers. BAPN is used as a positivecontrol.

FIG. 10 shows the effects of 5 compounds on LOX and LOL2 activity. Among5 candidates, 2 compounds (6232 and 6229) decreased the activity of boththe recombinant LOX (A) and LOXL2 (B) proteins, although LOXL2inhibition was weaker as compared to BAPN.

FIG. 11 shows the effect of 18 different 6232 analogs identified by SARanalysis on lysyl oxidase activity. The analogs were used at aconcentration of 5 μM, whereas BAPN was used at 10 mM.

FIG. 12 shows the effects of the short-listed analogs on lysyl oxidaseactivity and cell proliferation at multiple doses (2.5, 5, 10 and 20uM). BAPN was used at 10 mM concentration.

FIG. 13 shows Table 1, Structure-Activity Relationship of4′-methyl-N2-phenyl-[4,5′-bithiazole]-2,2′-diamine Lox Inhibitors.

FIG. 14 shows testing of the shortlisted analogs 6403, 6405, and 6415for toxicity in normal HUVEC, MCF12A and HFF cells. No significanteffect on the viability of normal cell lines was detected.

FIG. 15 shows in vivo toxicity testing for compound 6403. The upperpanel shows the body weight of the mice treated with different doses of6403, administered daily. The lower panel shows the blood cell countsfrom the vehicle vs. 200 mg/kg 6403-treated mice. No significant effecton body weight and blood counts were detected.

FIG. 16 shows the percentage growth inhibition in the TNBC cell line,MDA-MB-231, embedded in collagen and treated with the combination ofdoxorubicin and compounds 6232, 6403, 6405 and 6415.

FIG. 17 shows decrease in ECM crosslinking (as assessed by collagen(green) and fibronectin (red) staining) upon BAPN, 6232, 6403, 6405, or6415 treatments.

FIG. 18 shows the effects of the compound 6415 on LOX and LOXL2activity. 6415 was used at 5 and 10 uM concentrations in recombinantprotein based lysyl oxidase activity assay and inhibited the activity ofLOX and LOXL2 recombinant proteins. BAPN was used as a control at 10 mMdose.

FIG. 19 shows the lysyl oxidase activity and cell viability in Caki-1and ACHN cell lines treated with increasing doses of BAPN and 6232.

FIG. 20 shows the cellular morphology of ACHN and Caki-1 cell lines whentreated with 10 μM of the compound 6232.

FIG. 21 shows decrease in ECM crosslinking upon treatment of Caki-1cells with compound 6232 at 10 uM and BAPN at 10 mM.

FIG. 22 . Expression of LOX family members in cancer vs normal tissue byusing TCGA dataset. A. Pan-cancer expression of LOX family members inthe TCGA dataset. B. Expression of LOX, LOXL1 and LOXL2 in TNBCs vsnormal breast. C. Expression of LOX, LOXL1 and LOXL2 in pancreatictumors vs. normal tissue and kidney cancer vs normal tissue. ACC—Adenoidcystic carcinoma; BLCA—Bladder urothelial carcinoma; BRCA—Breast Cancer;CESC—Cervical squamous cell carcinoma; CHOL—Cholangiocarcinoma;COAD—Colon adenocarcinoma; DLBC-Diffuse large B cell lymphoma;ESCA—Esophageal cancer; GBM—Glioblastoma; HNSC—Head and neck squamouscell carcinoma; KICH—Kidney Chromophobe; KIRC—Kidney renal clear cellcarcinoma; KIRP—Kidney renal papillary cell carcinoma; LAML—AcuteMyeloid Leukemia; LGG—Low-grade glioma; LIHC—Liver HepatocellularCarcinoma; LUAD—Lung Adenocarcinoma; LUSC—Lung squamous cell carcinoma;MESO—Mesothelioma; OV—Ovarian Cancer; PAAD—Pancreatic adenocarcinoma;PCPG—Pheochromocytoma and paraganglioma; PRAD—Prostate Adenocarcinoma;READ—Rectum Adenocarcinoma; SARC—Sarcoma; SKCM—Skin cutaneous melanoma;STAD—Stomach adenocarcinoma; TGTC—Tenosynovial giant cell tumors;THCA—Thyroid Cancer; THYM—Thymoma; UCEC—Uterine Corpus EndometrialCarcinoma; UCS—Uterine Carcinosarcoma; UVM—Uveal Melanoma.

FIG. 23 . Association of the expression of LOX, LOXL1 and LOXL2 withsurvival in breast, pancreatic and kidney cancer patients. In the TCGAdataset, the expression of LOX, LOXL1 and LOXL2 associates with worseDFS in KIRC while higher expression of LOX and LOXL2 associates withworse relapse-free survival (RFS) in PAAD (in the TCGA dataset) and inchemotherapy-treated breast cancer (KM plotter database).

FIG. 24 . LOX expression is increased in doxorubicin resistance and itshigh expression is associated with worse survival in chemotherapytreated TNBC patients. A. LOX mRNA expression in sensitive vs. resistanttumor xenografts. B. Picrosirius staining of the tumors from A. C.Kaplan-Meier survival curve representing DFS in chemotherapy-treatedTNBC patients (n=77) separated from median LOX protein expression. D.IHC images of TNBC patient tissues with low and high LOX proteinexpression. *, P<0.05; **, P<0.01 in all figures.

FIG. 25 . LOX inhibition overcomes doxorubicin resistance in 3D culture.A, B. % growth inhibition of 3D collagen I-embedded MDA-MB-231 cellstreated with BAPN (A) or transfected with siLOX (B) in combination withdoxorubicin. C. IF staining of type I collagen (green) and fibronectin(red) in HFF-derived ECM incubated with vehicle or BAPN-treatedMDA-MB-231 cells. D. Relative doxorubicin fluorescence intensity uponBAPN-treatment in collagen I-embedded MDA-MB-231 cells. E. Western blotof LOX and FAK/Src signaling in collagen I-embedded MDA-MB-231 cellsupon doxorubicin (1 uM) and BAPN (5 mM) treatment for 24 h. F. Annexin Vstaining in 3D collagen I-embedded MDA-MB-231 cells treated withDoxo+BAPN combination from E. G. % growth inhibition of 3D collagenI-embedded MDA-MB-231 cells treated with doxorubicin in combination withFAK (500 nM) or Src (500 nM) inhibitors.

FIG. 26 . Inhibiting LOX overcomes resistance in acquired doxorubicinresistant MDA-MB-231 xenografts. A. Tumor growth in xenografts treatedwith low dose doxorubicin until resistance develops followed bytreatment with the combination of doxorubicin (2.5 mg/kg) and the LOXinhibitor, BAPN (100 mg/kg). B. Relative LOX activity in tumors from A.C. Representative images of Picrosirius red in tumors from A.

FIG. 27 . Targeting LOX in TNBC PDX organoids or tumors overcomesresistance. A. Scheme showing the selection pipeline of PDXs for in vivotesting. B. Correlation analysis of LOX mRNA expression with hypoxia andfocal adhesion scores in 15 different TNBC PDX models. Red dots show thepositions of the selected TM01278. C. Representative images of TM01278PDX organoids at day 0 and day 9 after treatment with doxorubicin (40nM) and BAPN (25 mM) treatment, alone or in combination (left) andquantification of organoid diameter upon combination therapy for 9 days(right) (n=12 (vehicle, Doxo, BAPN), n=11 (Doxo+BAPN)). D. Tumor growthof TM01278 PDX upon treatment with doxorubicin (2 mg/kg) and BAPN (100mg/kg), alone or in combination (n=5). Inset shows LOX expression in PDXtumors. E. Quantification of Picrosirius red staining in PDX tumors fromD (n=4). F. Doxorubicin fluorescence quantification in tumors from D(n=6). G. Western Blots of FAK/Src signaling in PDXs treated withdoxorubicin and BAPN alone or in combination.

FIG. 28 . Summary of our LOX or LOX family inhibitor discovery pipeline.

FIG. 29 . Characterization and selection of top hits from thehigh-throughput screen (HTS) of a diversified small-molecule library toidentify novel LOX inhibitors. A. Inhibition of cell-based LOX activityand cell viability upon treatment with top 20 candidate LOX inhibitorsat 10 uM. B. Doxorubicin sensitization upon combination of the selected5 compounds (5 uM) with doxorubicin (1 uM). C, D. LOX activity assaywith the recombinant LOX (C) LOXL2 (D) proteins incubated with 10 uM of6229 and 6232, and 10 mM BAPN. μM=microM.

FIG. 30 . LOX inhibitors generated based on SAR analysis.

FIG. 31 . Novel 6232 analogs, 6403 and 6415, effectively inhibit lysyloxidase activity, overcomes doxorubicin resistance and reduce collagencross-linking and fibronectin assembly. A. Inhibition of LOX activity inMDA-MB-231 cells treated with 5 μM of the LOX inhibitors. B. Doxorubicinsensitization with 6232 and its analogs, 6403 and 6415, in 3D collagen-Iculture. C. Collagen and fibronectin staining of vehicle or 6232 or 6403or 6415-treated MDA-MB-231 cells embedded in collagen I. BAPN is used asa positive control.

FIG. 32 . 6232 derivatives, 6403 and 6415, show no cytotoxicity innormal cells. Viability assay in normal breast cell line, MCF12A, humanforeskin fibroblast cell line, HFF and human endothelial cell line,HUVEC treated with 6403 or 6415.

FIG. 33 . While 6403 inhibits LOX recombinant protein, 6415 inhibitsboth LOX and LOXL2 recombinant proteins. A. Lysyl oxidase activity assaywith the recombinant LOX, and LOXL2 proteins in the presence or absenceof 10 uM 6403. B. Activity assay with the recombinant LOX, and LOXL2proteins in the presence or absence of 10 uM 6415.

FIG. 34 . 6232, 6403 and 6415 are more potent than LOX family inhibitor,BAPN. IC50 for cellular lysyl oxidase activity upon treatment ofMDA-MB-231 cells with different LOX inhibitors. uM=microM.

FIG. 35 . 6403 and 6415 led to doxorubicin chemosensitization in 3Dculture of TNBC cell lines. Doxorubicin sensitization with 6232 analogs,6403 and 6415, in 3D collagen-I culture of MDA-MB-231 (Doxo1: 0.75 uM,Doxo2: 1 uM, 6403: 5 μM, 6415:5 μM) (A) and HCC1143 (Doxo1: 1 uM, Doxo2:2 μM, 6403: 5 μM, 6415:5 μM) (B).

FIG. 36 . Combination of 6403 and 6415 with doxorubicin reduced thegrowth of the TNBC organoids. A. Representative images of TM01278 PDXorganoids at day 0 and day 9 after treatment with doxorubicin (50 nM)and 6403 and 6415 treatments, alone or in combination. B. Quantificationof organoid diameter and viability upon combination therapy for 9 days.

FIG. 37 . LOX inhibitors, 6232, 6403 and 6415, reduced the migrationcapacity of the TNBC cell lines. RTCA-CIM plate used to measure thereal-time migration of MDA-MB-231 cells upon treatment with 20 μM of6232, 6403 and 6415 for 48 hours. (* p<0.05; ** p<0.01)

FIG. 38 . Dose dependent inhibition of cellular LOX activity and growthinhibition in 3D upon treatment with 6232 and its analogs, 6403 and6415.in ACHN cancer cell line. Inhibition of cell-based LOX activity inACHN (A) and growth inhibition in collagen:matrigel (1:1) culture (B)cancer cell lines upon treatment with LOX inhibitors 6232, 6403 and 6415(uM range). BAPN is used as a positive control (mM range) in 3D cellviability assay. Growth inhibition was measured by using 3D Cell TiterGlo kit.

FIG. 39 . Inhibition of cellular LOX activity in pancreatic and kidneycancer cell lines. Inhibition of cellular LOX activity upon thetreatment with BAPN, 6232, 6403 and 6415 in kidney cell line Caki-1 (A)and pancreatic cancer cell lines Mia-Paca-2 (B) and PANC1 (C).

FIG. 40 . Treatment with 6232, and its analogs 6403 and 6415 inhibitedthe TGF-β-induced fibrosis in HFF cells. 24 hours serum-starved HFFcells were pretreated with inhibitors 1 hours. Then, fibrosis wasinduced by 10 uM of TGF-β for 48 hours in HFF cells. ALK5 (10 μM)inhibitor is used as a negative control. LOX inhibitors 6232, 6403 and6415 were used at 10 uM concentration. HFF cells were stained withalpha-SMA and fibronectin primary antibodies. Nucleus of the cells waslabeled with DAPI.

FIG. 41 . In vivo testing of 6403 with respect to PK and toxicity. A.Summary of PK parameters of 6403 given orally with 50 mg/kg, n=3. B. H&Estaining of organs of BALB/c mice treated daily with 50 or 200 mg/kg of6403 for one week. C. Body weight of mice treated with vehicle or 25,50, 75, 100 or 200 mg/kg of 6403 for 5 days. D. Cell-free enzymaticMAO-A and MAO-B assay.

FIG. 42 . Efficacy of 6403 as a single agent in ccRC xenografts. Tumorvolume (A), % body weight change (B) and blood count (C) of 6403-treated(50 mg/kg, PO, daily) ACHN xenografts. μg=microgram. LOX expression inACHN cells is provided by an inset next to the graph in A.

FIG. 43 . 6403 overcomes doxorubicin resistance without major toxicityin a syngeneic mouse model, 4T1. A. Tumor growth in 4T1 model treatedwith doxorubicin (2 mg/kg, I.V, weekly), 6403 (50 mg/kg, P.O, daily) ortheir combination. Representative pictures of the tumors shown on theright panel. B. Western blot analysis of LOX in 4T1 tumors from A. C. %body weight change of mice from A. D. Blood cell counts in mice from Aat the end of the experiment.

Method

Cell Culture and Reagents

Human TNBC cell lines, MDA-MB-231, and HCC1143; Pancreatic cancer celllines, PANC1, and Mia-PACA-2; Kidney cancer cell lines, ACHN, andCaki-1. All the cells were cultured in Dulbecco Modified Eagle Medium(Gibco, USA) supplemented with 50 U/ml penicillin/streptomycin, 1%non-essential amino acids and 10% fetal bovine serum (Gibco, USA). Thecell lines were authenticated and tested for mycoplasma contaminationregularly using MycoAlert mycoplasma detection kit (Lonza, NJ, USA). Thecumulative culture length of cells between thawing and use in this studywas less than 20 passages.

Western Blotting

Protein isolation and Western blotting were done as previouslydescribed, see Mishra, R. R. et al. Reactivation of cAMP Pathway byPDE4D Inhibition Represents a Novel Druggable Axis for OvercomingTamoxifen Resistance in ER-positive Breast Cancer. Clinical CancerResearch: An Official Journal of The American Association for CancerResearch 24, 1987-2001, doi:10.1158/1078-0432.CCR-17-2776 (2018) andSaatci, O. et al. Targeting PLK1 overcomes T-DM1 resistance viaCDK1-dependent phosphorylation and inactivation of Bcl-2/xL inHER2-positive breast cancer. Oncogene 37, 2251-2269,doi:10.1038/s41388-017-0108-9 (2018). Briefly, RIPA buffer was used toisolate total protein lysate in the presence of protease and phosphataseinhibitor cocktails, and protein concentrations were measured using theBCA Protein Assay Reagent Kit (Thermo Fisher Scientific, MA, USA). Equalamounts of protein were separated using 8-10% SDS-PAGE gel. Separatedproteins were transferred to PVDF membranes (Bio-Rad, CA, USA) using aTrans-Blot turbo transfer system (Bio-Rad, CA, USA) and incubated withprimary antibodies (LOX (Novus), LOXL1 (Santa-Cruz) and LOXL2 (Abcam)).Horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies(Cell signaling Technology, MA, USA) were used as secondary antibodies,and signals were detected by enhanced chemiluminescence (Thermo FisherScientific, MA, USA). Images were acquired using Image Lab Software(Biorad, CA, USA).

Cell Embedding in Type I Collagen:Matrigel (1:1) Mix

Collagen solution was prepared at a concentration of 0.5-1 mg/ml fromthe rat tail collagen I (Corning, USA) with a neutralization stepincluding the addition of 1N NaOH solution. Cells were trypsinazed andresuspended in collagen:matrigel mix (1:1). 8×10³ cells were seeded into96-well or μ-Slide 8-well glass bottom chambers. After 1 hour ofincubation at room temperature, media was added on top of the solidifiedcollagen:matrigel mix. Drug treatments were done 12 hours after cellseeding.

Immunofluorescence Staining

Immunofluorescence staining of cells was done in μ-Slide 8-well glassbottom chambers (Ibidi, Germany). Cells were fixed with 4%paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 for 10min and blocked in 5% BSA-PBS. Samples were incubated with primary(Collagen I, Fibronectin, F-actin, a-SMA) and secondary Alexa Fluor 647or 488-labeled antibodies for 1.5 hours at room temperature. Cells werealso counterstained with DAPI for 5 min. Images were acquired using ZEN2012 SP5 (Black) LSM 700 (Carl Zeiss, DE).

Lysyl Oxidase Activity Assay

For cell-based lysyl oxidase activity, fluorometric Lysly OxidaseActivity Assay Kit (Abcam, USA) was used according to manufacturer'sinstructions. Briefly, after the treatment, 50 μl of supernatant fromeach well was incubated with reagent mix and fluorescence measured inmultimode-reader at Ex/Em=540/590 nm wavelength.

For in vitro lysyl oxidase activity (for recombinant proteins), LysylOxidase Activity kit (Biovision, USA) was used according tomanufacturer's instructions. Briefly, 1 μg of LOX (Novus, USA), 0.2 μgof LOXL1 (Abcam, USA) and 0.1 μg of LOXL2 (Abcam, USA) were incubatedwith the reagent mix in the presence of inhibitors or vehicle. Lysyloxidase activity of the wells was measured in multimode reader (every 30sec, total 2.5 hours) in luminescence mode.

Chemosensitization in 3D Culture

Cells were embedded in type I collagen:matrigel mix as described above.Cells were grown in the presence of doxorubicin or LOX inhibitors aloneor combination of LOX inhibitors with doxorubicin for 48 hours. Afterthe treatment, cell viability was measured by using 3D Cell Titer Glo(Promega, USA) kit.

PDX-Derived TNBC Organoids

TNBC organoids were established from a fresh surgical tissue by cuttingthe tumor into small pieces and incubating in collagenase A solutionwith ROCK inhibitor on a shaker at 37° C. for 30 minutes. Thecollagenase activity was inhibited by adding FBS, and pipetting was doneto ensure the formation of almost a single cell solution. After severalwashes with PBS, the cell pellet was dissolved in matrigel. Breastorganoid media containing ROCK and GSK inhibitors was added after theMatrigel solidified. See, Sachs, N. et al. A Living Biobank of BreastCancer Organoids Captures Disease Heterogeneity. Cell 172, 373-386 e310,doi:10.1016/j.cell.2017.11.010 (2018). For drug testing studies,organoids were disrupted to single cells by digesting at 37° C. for 30mins with TrypLE (A1217701, Gibco, NY, USA) in the presence of 10 μMRock inhibitor (s1049, Selleckchem, TX, USA). Organoids were plated intowells of 96 well plate (20.000 cells/well) on a collagen-coated surfacewith media containing 2% Matrigel (356252, Corning, N.Y., USA). Drugswere added 72 hours after plating. Cells were grown in the presence ofdrug or vehicle for 9 days, and photos were taken from each well to showthe organoid size. Organoid viability was measured by using 3D CellTiter Glo (Promega, USA) kit.

Migration Assay

RTCA CIM Plate was used to assess the migration capacity of the cellsupon LOX inhibitor treatment according to manufacturer's instructions.Briefly, 160 μl of 10% FBS containing media was added into each bottomchamber/50 μl of FBS free media into upper chamber and incubated 1 hourat 37° C. After the background measurement, 5×10⁴ cells were seeded inFBS-free media into upper chamber in the presence of LOX inhibitors orvehicle. After the 30 min incubation at RT, real time cell migration wasrecorded for 48 hours.

Fibrosis Assay

Human foreskin fibroblasts (HFF), see Li, M. et al. Icaritin inhibitsskin fibrosis through regulating AMPK and Wnt/β-catenin signaling. CellBiochemistry and Biophysics 79, 231-238 (2021), cells were seeded intoμ-Slide 8-well glass bottom chambers (Ibidi, Germany). Next day, mediaof the cells were replaced with 0.5% FBS contain media and incubatedwith 24 hours. Then, cells were pretreated either with ALK5 inhibitor(Sellekchem, USA) or LOX inhibitors for 1 hour and cells were incubatedwith 10 ng/ml of TGF-beta (R&D Biosystems, USA) for 48 hours. Stainingof the markers followed the same procedure described in theImmunofluorescence section above.

PK Analysis

PK profiles of lead LOX inhibitor, 6403 was tested in BALB/c mice. Threemice per group were dosed by oral gavage at 50 mg/kg. Serum samples werecollected at time points; 0.25, 0.5, 2, 7 and 24 hours. 6403 was dilutedwith serum from healthy mice as standard and serum levels of LOXinhibitors were quantified by LC-MS/MS methods, compared to the standardreadings and followed by calculation of PK parameters, includingC_(max), AUC, T_(1/2) values, clearance (Cl) and oral bioavailability (F%). C_(max) represents the maximum concentration of drug that observedin the serum. AUC is a measure of the total exposure of the drug in theserum, T_(1/2), half life, is the time required to eliminate half of thedrug. Cl is defined as the volume of blood from which all drug isremoved per minute.

MAO-GLO Assay

MAO-GLO assay (Promega, USA) was used to measure the MAO-A and MAO-Bactivity upon LOX inhibitor treatment according to the manufacturer'sinstructions. For the enzyme activity, substrate was used at aconcentration of 160 μM and 16 μM for MAO-A and MAO-B, respectively.MAO-A and MAO-B enzymes (Active Motif, USA) were used in 0.2 μg/perwell. Reaction mix, enzymes, and drugs (30, 10, 3, 1, 0.3, and 0.1 nM)were mixed and incubated for 3 hours at RT. Then, 50 μl of luciferin wasadded into wells and incubated for 20 min at RT. Luciferase signals weremeasured by using a multimode reader.

Syngeneic and Xenograft Mice Tumor Model

For kidney xenografts, 4×10⁶ ACHN cells were mixed with 1:1 DMEM andmatrigel and injected into the flank of the BALB/c nude mice. Mouseweight and tumor volumes were measured every second day using calipers.When tumor sizes reach 100 mm³, mice were randomly distributed intovehicle and 6403 treatment group. Mice were treated daily by oral gavagewith vehicle v (50% PEG400, 1% Tween 80 in acetate buffer pH=4) or 50mg/kg of 6403.

500,000 4T1 cells were injected into MFP of 6-8 weeks old female BALB/cmice. When the tumors reach 100 mm3, mice were randomly distributed to 4groups and treated with vehicle, doxorubicin (2 mg/kg once a week,I.V.), 6403 (50 mg/kg daily, oral gavage) or the combination ofdoxorubicin with 6403. Tumor volumes were measured using a caliper everytwo days, and body weights were also recorded. After 3 weeks oftreatment, blood was withdrawn, and blood cells were quantified usingVetScan HM5 Hematology Analyzer.

Bioinformatic Analyses

Patient data for normal vs. tumor comparisons were retrieved from theNCBI GEO database (accession ID: GSE76250). Patient data for survivalanalyses were retrieved from TCGA database using cBio Portal, seeCerami, E. et al. Vol. 2 401-404 (AACR, 2012) and Lopes, C. T. et al.Cytoscape Web: an interactive web-based network browser. Bioinformatics26, 2347-2348 (2010), and from online survival analysis tool,KM-plotter. See, Gyorffy, B. et al. An online survival analysis tool torapidly assess the effect of 22,277 genes on breast cancer prognosisusing microarray data of 1,809 patients. Breast Cancer Research andTreatment 123, 725-731 (2010). Survival curves were generated based on25^(th) percentile separation using Kaplan-Meier method, andsignificance between groups was calculated by Log-rank test.Significance between normal vs tumor expression was calculated usingunpaired Student's t-test. Boxplots show median number, and the 25^(th)to 75^(th) quartiles. Upper and lower whiskers denote the minimum andmaximum values in the corresponding group. Graphs and curves were drawnusing GraphPad software (GraphPad software Inc., La Jolla, Calif., USA).Significance threshold was described as *P<0.05; **P<0.01; ***P<0.001;ns, not significant.

The compounds of the current disclosure have wide-ranging impact ondisease treatment. Indeed, the compounds disclosed herein, and or theirpharmaceutically acceptable salts, may be administered in atherapeutically effective amount to inhibit LOX and LOX family membersin both a LOX-specific as well as pan-LOX manner. Indeed, the compoundsdisclosed herein may be administered as anti-cancer agents where canceris selected from the group consisting of lung cancer; breast cancer;colorectal cancer; anal cancer; pancreatic cancer; prostate cancer;ovarian carcinoma; liver and bile duct carcinoma; esophageal carcinoma;non-Hodgkin's lymphoma; bladder carcinoma; carcinoma of the uterus;glioma, glioblastoma, medullablastoma, and other tumors of the brain;myelofibrosis, kidney cancer; cancer of the head and neck; cancer of thestomach; multiple myeloma; testicular cancer; germ cell tumor;neuroendocrine tumor; cervical cancer; oral cancer, carcinoids of thegastrointestinal tract, breast, and other organs; signet ring cellcarcinoma; mesenchymal tumors including sarcomas, fibrosarcomas,haemangioma, angiomatosis, haemangiopericytoma, pseudoangiomatousstromal hyperplasia, myofibroblastoma, fibromatosis, inflammatorymyofibroblastic tumour, lipoma, angiolipoma, granular cell tumour,neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma,osteosarcoma, leiomyoma or a leiomysarcoma.

Indeed, compounds 6232, 6403, and 6415, however the current disclosureis not limited to just these compounds and should not be readrestrictively or exclusively in this sense, can be used to targetcancers and diseases caused by LOX family members as well as to inducemetastasis inhibition. The compounds disclosed herein inhibit bothcell-based lysyl oxidase activity and recombinant protein based lysyloxidase activity. The compounds may a pharmaceutically acceptable saltthereof, or a pharmaceutical composition, used in the treatment offibrosis, neurodegenerative, angiogenesis-related diseases and cancer,which show aberrant LOX family expression and/or lysyl oxidase activity.Indeed, the compounds of the current disclosure may be used as a singleagent and/or in combination with standard of care therapies, as well aschemotherapy, immunotherapy or radiotherapy in both adjuvant andneo-adjuvant settings. Further, the compounds of the current disclosuremay be used with small molecule treatments to address thediseases/disorders herein. The compounds disclosed herein may be used totreat diabetes-induced fibrosis and retinopathy, Fibrosis may includeliver fibrosis, pulmonary fibrosis, renal fibrosis, myocardial fibrosis,cystic fibrosis, idiopathic pulmonary fibrosis, radiation-inducedfibrosis, ocular fibrosis, Peylony's disease and scleroderma, orrespiratory disorders, abnormal wound healing and repair, postoperativesurgery, cardiac arrest-related fibrosis, excess or abnormal depositionof fibrotic material, all associated with disorders such as Crohn'sdisease and inflammatory bowel disease, liver, kidney and pancreasfibrosis. The compounds can also be used to treat angiogenesis relateddiseases. Indeed, the compounds disclosed herein may be administeredwith or in conjunction with a second therapeutic agent selected fromanti-cancer agent, anti-inflammatory, anti-hypertensive, anti-fibrotic,anti-angiogenic agent, and an immunosuppressive agent. The currentcompounds also may be used to treat kidney disorders such as kidneyfibrosis, renal fibrosis, acute kidney injury, chronic kidney disease,diabetic nephropathy, glomerulosclerosis, vesicoureteral reflux,tubulointerstitial renal fibrosis and glomerulonephritis. Further, thecompounds of the current disclosure may be directed to healing,remedying hypertrophic scarring, keloids, and diabetic skin and may beused for cosmetic applications. See Pathol Biol (Paris) 2005 September;53(7):448-56. doi: 10.1016/j.patbio.2004.12.033.

Examples Synthesis Routes

Compounds 6232, 6398, 6399, 6400, 6401, 6402, 6403, 6404, 6405, 6406,6407, 6408, 6409, 6410, 6411, 6412, 6413, 6414, and 6415 were purchasedfrom Chembridge Corporation (San Diego, Calif., USA, 92121) and testedas is. The synthesis of compounds 6424 (synthesized as formate salt of6403), 6432 (synthesized as formate salt of 6405), 6433 (synthesized asformate salt of 6232) and 6439 (synthesized as formate salt of 6415) aredescribed below. Compounds 6425 and 6426 have not been previouslysynthesized and are novel. Compounds purchased from Chembridge can beresynthesized using the same general methods. The general scheme isshown below including the synthesis of future derivatives includingsubstitutions on the 4-methylthiazol-2-amine and aniline rings:

Synthesis of 1-(2-amino-4-methyl-thiazol-5-yl)-2-bromo-ethanone (1)

1-(2-amino-4-methyl-thiazol-5-yl)ethanone (5 g) was dissolved in HBr (20ml) and allowed to warm to 60° C. Br₂ (4.6 g) was then dissolved in1,4-dioxane and was added in a dropwise manner. The reaction was allowedto stir at 60° C. for 3 hours. After cooling to RT, the mixture wasbasified with Na₂CO₃ after which solid product came out of solution. Thesolid was removed by filtration. After extraction of the remainingsolution with ethyl acetate, the organic layer was then washed withbrine, dried over sodium sulfate, and was rotovapped to dryness. Theremaining crude material was combined with the filtered solid and wasdried in a vacuum desiccator (7.5 g, 50% purity), ESI-MS (pos): m/z 235(M+H)⁺ (expected 235.10).

Synthesis ofN-[4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]amino]phenyl]acetamide(6403, 6424) Synthesis of N-[4-(carbamothioylamino)phenyl]acetamide

N-(4-aminophenyl)acetamide (6 g) was dissolved in anhydrous DCM andbenzoyl isothiocyanate (8.15 g) was added in a dropwise fashion at RT.The reaction was then allowed to stir overnight. The solvent was removedand the products re-dissolved in 1:1 THF/1M NaOH, and then allowed toreflux at 75° C. for 3 hours. The sample was cooled to RT, diluted inwater (˜150 mL) and was extracted ethyl acetate (4×) to yieldN-[4-(carbamothioylamino)phenyl]acetamide (2.2 g, 23% yield, 86% purity)ESI-MS (pos): m/z 210.0309 (M+H)⁺ (expected 210.06).

Synthesis ofN-[4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]amino]phenyl]acetamide(6403, 6424)

N-[4-(carbamothioylamino)phenyl]acetamide (1.1 g) and1-(2-amino-4-methyl-thiazol-5-yl)-2-bromo-ethanone (1.85 g) weredissolved in 30 mL ethanol and the solution was heated to 90° C. for 2-3h. After completion of the starting material the solvent was removedunder reduced pressure at 45° C. water bath temperature. The crudematerial was re-dissolved in 3 mL of DMSO and purified by MediumPressure Liquid Chromatography using a C18 reverse phase column andacetonitrile-water as eluent with 1% aqueous formic acid additive toobtain the desired product (1.3 g, 99% purity) in good yield (71.6%).¹H-NMR (400 MHz, D₆ DMSO) δ ppm 10.20 (s, 1H), 9.849 (s, 1H), 8.141 (s,1H), 7.500-7.731 (m, 6H), 2.353 (s, 3H), 2.025 (s, 3H). ESI-MS (pos):m/z 346.1247 (M+H)⁺ (expected 346.08).

Synthesis of4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]-methyl-amino]phenol(6415, 6439) 1) Synthesis of 4-(methylamino)phenol

An oven dried 250 mL round bottom flask was charged with 4-aminophenol(5 g), and to 20 mL of dimethylcarbonate (20 ml) was added followed bydimethoxyethane (20 mL) and NaY catalyst (5 g) in a sequential manner.The mixture was heated to 86° C. for 16 h. Then the solvent and excessreagent was evaporated and the crude material re-dissolved in 70-80 mLof Methanol and solid catalyst was filtered through celite pad and themethanol was concentrated under reduced pressure at 45° C. water bathtemperature to obtained the product (5.6 g, yield 14%). ESI-MS (pos):m/z 124 (M+H)⁺. Expected 124.

2) Synthesis of 1-(4-aminophenyl)-1-methylthiourea

Crude 4-(methylamino)phenol (2.5 g) was dissolved in anhydrous DCM, thenthe Benzoyl isothiocyanate (5.0 g) was added in a dropwise fashion atroom temperature (25° C.). During the process of addition the reactionmixture was slightly warmed. The reaction was then allowed to stir atroom temperature overnight. After 16 h, the DCM was evaporated todryness, then a 1:1 THF/1M NaOH solution was added until the solids werecompletely dissolved. The reaction was then allowed to reflux at 75° C.for 8 hr. The organic and aqueous layers were separated, aqueous layerwas acidified with conc. HCl (pH=3-4) and then extracted with ethylacetate, the solids were filtered and the organic layer was concentratedunder reduced pressure. LCMS showed the desired mass peak at 183.

3) Synthesis of4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]-methyl-amino]phenol(6415, 6435)

1-(4-aminophenyl)-1-methylthiourea (1.8 g) and1-(2-amino-4-methyl-thiazol-5-yl)-2-bromo-ethanone (3.48 g) weredissolved in 30 mL ethanol and the solution was heated to 90° C. for 2-3h. After completion of the starting material the solvent was removedunder reduced pressure at 45° C. water bath temperature. The crudematerial was re-dissolved in 3 mL of DMSO and purified by MediumPressure Liquid Chromatography using a C₁₈ reverse phase column andacetonitrile-water as eluent with 1% aqueous formic acid additive toobtain the desired product (2.5 g, 99% purity) in good yield (79.5%).¹H-NMR (400 MHz, D₆ DMSO) δ ppm 7.235-7.258 (m, 2H), 6.900 (s, 1H),6.818-6.850 (m, 2H), 3.379 (s, 3H), 2.2848 (s, 3H). ESI-MS (pos): m/z319.1 (M+H)⁺ (expected 319.1).

Synthesis ofN-[4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]amino]phenyl]-3-methoxy-propanamide(6425) 1) Synthesis of N-[4-(carbamothioylamino)phenyl]-3-methoxy-propanamide

N-(4-aminophenyl)-3-methoxy-propanamide (0.24 g) was suspended in 2 mLof dry DCM and the benzoyl isothiocyanate (0.26 g) was separatelydissolved in 1 mL of dry DCM. The benzoyl isothiocyanate was then addedto the aniline dropwise over the course of 1 min with stirring. Thesolution was then rotovapped to dryness and dissolved in a solution ofTHF/1M NaOH (1:1). The solution was then refluxed at 75° C. for 3 hours.The sample was then cooled to room temperature, diluted with water, andextracted 3× with ethyl acetate. The organic solution was then driedover sodium sulfate, filtered, and rotovapped to dryness and purifiedwith medium pressure liquid flash chromatography to yield the product(0.053 g, 16.6% yield, 99% purity). ESI-MS (pos): m/z 254.1 (M+H)⁺(expected 254.1).

2) Synthesis ofN-[4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]amino]phenyl]-3-methoxy-propanamide(6425)

N-[4-(carbamothioylamino)phenyl]-3-methoxy-propanamide (0.019 g) and 1(0.023 g) were dissolved in ethanol and were allowed to reflux for 90min after which the sample was then rotovapped to dryness, dissolved inDMSO, and was purified by reverse-phase flash chromatography to yieldthe product (0.029 g, 99% purity). ESI-MS (pos): m/z 390.1 (M+H)⁺(expected 390.1).

Synthesis ofN-[4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]amino]phenyl]pentanamide(6526) 1) Synthesis of N-[4-(carbamothioylamino)phenyl]pentanamide

N-(4-aminophenyl)pentanamide (0.044 g) was suspended in 2 mL of dry DCMand the benzoyl isothiocyanate (0.047 g) was separately dissolved in 1mL of dry DCM. The benzoyl isothiocyanate was then added to the anilinedropwise over the course of 1 min with stirring. The solution was thenrotovapped to dryness and dissolved in a solution of THF/1M NaOH (1:1).The solution was then refluxed at 75° C. for 3 hours. The sample wasthen cooled to room temperature, diluted with water, and extracted 3×with ethyl acetate. The organic solution was then dried over sodiumsulfate, filtered, and rotovapped to dryness and purified with mediumpressure liquid flash chromatography to yield the product (0.007 g).ESI-MS (pos): m/z 252.1 (M+H)⁺ (expected 252.1).

2) Synthesis ofN-[4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]amino]phenyl]pentanamide(6526)

N-[4-(carbamothioylamino)phenyl]pentanamide (0.0073 g) and 1 (0.0047 g)were dissolved in ethanol and were allowed to reflux for 90 min afterwhich the sample was then rotovapped to dryness, dissolved in DMSO, andwas purified by reverse-phase flash chromatography to yield the product(0.038 g, 99% purity). ESI-MS (pos): m/z 388.1 (M+H)⁺ (expected 388.1).

Synthesis of 4-methyl-5-[2-(4-nitroanilino)thiazol-4-yl]thiazol-2-amine(6232, 6433) 1) Synthesis of (4-nitrophenyl)thiourea

4-nitroaniline (2.0 g) was suspended in 2 mL of dry DCM and the benzoylisothiocyanate (2.95 g) were separately dissolved in 1 mL of dry DCM.The benzoyl isothiocyanate was then added to the aniline dropwise overthe course of 1 min with stirring. The solution was then rotovapped todryness and dissolved in a solution of THF/1M NaOH (1:1). The solutionwas then refluxed at 75° C. for 1 hour. The sample was then cooled toroom temperature, diluted with water, and extracted 3× with ethylacetate. The organic solution was then dried over sodium sulfate,filtered, and rotovapped to dryness and purified with medium pressureliquid flash chromatography to yield the product (0.181 g). ESI-MS(pos): m/z 198.0 (M+H)+(expected 198.0).

2) Synthesis of4-methyl-5-[2-(4-nitroanilino)thiazol-4-yl]thiazol-2-amine (6232, 6433)

(4-nitrophenyl)thiourea (0.18 g) and 1 (0.30 g) were dissolved inethanol and were allowed to reflux for 90 min after which the sample wasthen rotovapped to dryness, dissolved in DMSO, and was purified byreverse-phase flash chromatography to yield the product (0.041 g, 99%purity). (expected 334.0). ¹H-NMR (400 MHz, D₃ Methanol) 6 ppm8.027-8.124 (m, 3H), 7.742-7.766 (d, 2H), 6.667 (s, 1H), 2.326 (s, 3H).ESI-MS (pos): m/z 334.0 (M+H)⁺ expected 334.0.

Synthesis of4-((2′-amino-4′-methyl-[4,5′-bithiazol]-2-yl)amino)benzenesulfonamide(6405, 6432) 1) Synthesis of (4-sulfamoylphenyl)thiourea

4-aminobenzenesulfonamide (0.10 g) was dissolved in HCl with warming andwas then allowed to cool to RT. Potassium thiocyanate (0.056 g) was thenadded and the sample irradiated in the microwave at 90° C. for 2 hours.The sample was then transferred to a vial, chilled on ice and scratchedto precipitate, and was filtered, leaving a white solid (0.006 g, 99%pure) ESI-MS (pos): m/z 232.0 (M+H)⁺ (expected 232.3).

2) Synthesis of4-((2′-amino-4′-methyl-[4,5′-bithiazol]-2-yl)amino)benzenesulfonamide(6405, 6432)

(4-sulfamoylphenyl)thiourea (0.13 g) and 1 (0.12 g) were dissolved inethanol and were allowed to reflux for 90 min after which the sample wasthen rotovapped to dryness, dissolved in DMSO, and was purified byreverse-phase flash chromatography to yield the product (0.064 g, 99%purity). ESI-MS (pos): m/z 368.0 (M+H)⁺ (expected 368.0).

Various modifications and variations of the described methods,pharmaceutical compositions, and kits of the disclosure will be apparentto those skilled in the art without departing from the scope and spiritof the disclosure. Although the disclosure has been described inconnection with specific embodiments, it will be understood that it iscapable of further modifications and that the disclosure as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out thedisclosure that are obvious to those skilled in the art are intended tobe within the scope of the disclosure. This application is intended tocover any variations, uses, or adaptations of the disclosure following,in general, the principles of the disclosure and including suchdepartures from the present disclosure come within known customarypractice within the art to which the disclosure pertains and may beapplied to the essential features herein before set forth.

What is claimed is:
 1. A method for inhibiting lysyl oxidasescomprising: administering a therapeutically effective amount of acompound or its pharmaceutically acceptable salt having formula I;

Wherein: R1 is CH₃; R₂ is H, NH₂, or NHR; R₃ is H or CH₃; R₄ is H; R₅ isH; R₆ is NHCOCH₃, NO₂, C₂OH, or OH; R₇ is H; Q1 is N; Q2 is S; Q3 is N;Q4 is CH; Q5 is S; Q6 is CH; Q7 is CH; Q8 is C; and Q9 is CH; whereinthe compound or its pharmaceutically acceptable salt inhibits cell-basedlysyl oxidase activity and/or recombinant protein based lysyl oxidaseactivity assays.
 2. The method of claim 1, wherein the therapeuticallyeffective amount of the compound or its pharmaceutically acceptable saltis administered to treat a neurodegenerative disease, anangiogenesis-related disease, Alzheimer's disease, fibrosis including:liver fibrosis, pulmonary fibrosis, renal fibrosis, myocardial fibrosis,cystic fibrosis, idiopathic pulmonary fibrosis, radiation-inducedfibrosis, ocular fibrosis, Peylony's disease and scleroderma, orrespiratory disorders, abnormal wound healing and repair, postoperativesurgery, cardiac arrest-related fibrosis, excess or abnormal depositionof fibrotic material, all associated with disorders such as Crohn'sdisease and inflammatory bowel disease, liver, kidney and pancreasfibrosis, diabetes, cerebral hemorrhage with amyloidosis, cardiachypertrophy, Hutchinson-Gilford Progeria Syndrome, retinopathy,chemoresistance, and/or a kidney disorder including: kidney fibrosis,renal fibrosis, acute kidney injury, chronic kidney disease, diabeticnephropathy, glomerulosclerosis, vesicoureteral reflux, hypertrophicscarring, keloids, diabetic skin, tubulointerstitial renal fibrosisand/or glomerulonephritis.
 3. The method of claim 2, wherein the canceris selected from the group comprising lung cancer; breast cancer;colorectal cancer; anal cancer; pancreatic cancer; prostate cancer;ovarian carcinoma; liver and bile duct carcinoma; esophageal carcinoma;non-Hodgkin's lymphoma; bladder carcinoma; carcinoma of the uterus;glioma, glioblastoma, medullablastoma, and other tumors of the brain;myelofibrosis, kidney cancer; cancer of the head and neck; cancer of thestomach; multiple myeloma; testicular cancer; germ cell tumor;neuroendocrine tumor; cervical cancer; oral cancer, carcinoids of thegastrointestinal tract, breast, and other organs; signet ring cellcarcinoma; mesenchymal tumors including sarcomas, fibrosarcomas,haemangioma, angiomatosis, haemangiopericytoma, pseudoangiomatousstromal hyperplasia, myofibroblastoma, fibromatosis, inflammatorymyofibroblastic tumour, lipoma, angiolipoma, granular cell tumour,neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma,osteosarcoma, leiomyoma and/or a leiomysarcoma.
 4. The method of claim1, wherein the compound or its pharmaceutically acceptable salt hasformula:


5. The method of claim 1, wherein the compound or its pharmaceuticallyacceptable salt has formula:


6. The method of claim 1, wherein the compound or its pharmaceuticallyacceptable salt has formula:


7. The method of claim 1, wherein the compound or its pharmaceuticallyacceptable salt has formula:


8. The method of claim 1, wherein the therapeutically effective amountof a compound or its pharmaceutically acceptable salt is employed as ananti-cancer agent wherein cancer is selected from the group comprisinglung cancer; breast cancer; colorectal cancer; anal cancer; pancreaticcancer; prostate cancer; ovarian carcinoma; liver and bile ductcarcinoma; esophageal carcinoma; non-Hodgkin's lymphoma; bladdercarcinoma; carcinoma of the uterus; glioma, glioblastoma,medullablastoma, and other tumors of the brain; myelofibrosis, kidneycancer; cancer of the head and neck; cancer of the stomach; multiplemyeloma; testicular cancer; germ cell tumor; neuroendocrine tumor;cervical cancer; oral cancer, carcinoids of the gastrointestinal tract,breast, and other organs; signet ring cell carcinoma; mesenchymal tumorsincluding sarcomas, fibrosarcomas, haemangioma, angiomatosis,haemangiopericytoma, pseudoangiomatous stromal hyperplasia,myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumour,lipoma, angiolipoma, granular cell tumour, neurofibroma, schwannoma,angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyomaand/or a leiomysarcoma.
 9. The method of claim 1, wherein thetherapeutically effective amount of the compound or its pharmaceuticallyacceptable salt is administered to reduce cardiotoxicity side effects ofchemotherapy and/or immunotherapy.
 10. The method of claim 1, whereinthe therapeutically effective amount of the compound or itspharmaceutically acceptable salt is administered as a single agent or incombination with chemotherapy, immunotherapy and/or radiotherapy in bothadjuvant and neo-adjuvant settings.
 11. The method of claim 1, whereinthe therapeutically effective amount of the compound or itspharmaceutically acceptable salt is administered to induce metastasisinhibition.
 12. The method of claim 1, wherein the therapeuticallyeffective amount of the compound or its pharmaceutically acceptable saltis administered with a second therapeutic agent selected from ananti-cancer agent, an anti-inflammatory agent, an anti-hypertensiveagent, an anti-fibrotic agent, an anti-angiogenic agent, and/or animmunosuppressive agent.
 13. A compound for inhibiting lysyl oxidaseshaving formula:


14. The compound of claim 13 or its pharmaceutically acceptable saltadministered in a therapeutically effective amount to treat aneurodegenerative disease, an angiogenesis-related disease, Alzheimer'sdisease, fibrosis including: liver fibrosis, pulmonary fibrosis, renalfibrosis, myocardial fibrosis, cystic fibrosis, idiopathic pulmonaryfibrosis, radiation-induced fibrosis, ocular fibrosis, Peylony's diseaseand scleroderma, or respiratory disorders, abnormal wound healing andrepair, postoperative surgery, cardiac arrest-related fibrosis, excessor abnormal deposition of fibrotic material, all associated withdisorders such as Crohn's disease and inflammatory bowel disease, liver,kidney and pancreas fibrosis, diabetes, cerebral hemorrhage withamyloidosis, cardiac hypertrophy, Hutchinson-Gilford Progeria Syndrome,retinopathy, chemoresistance, and/or a kidney disorder including: kidneyfibrosis, renal fibrosis, acute kidney injury, chronic kidney disease,diabetic nephropathy, glomerulosclerosis, hypertrophic scarring,keloids, diabetic skin, vesicoureteral reflux, tubulointerstitial renalfibrosis and/or glomerulonephritis.
 15. The compound of claim 13 or itspharmaceutically acceptable salt administered as a single agent or incombination with chemotherapy, immunotherapy and/or radiotherapy in bothadjuvant and neo-adjuvant settings.
 16. The compound of claim 13 or itspharmaceutically acceptable salt administered with a second therapeuticagent selected from an anti-cancer agent, an anti-inflammatory agent, ananti-hypertensive agent, an anti-fibrotic agent, an anti-angiogenicagent, and/or an immunosuppressive agent.
 17. A compound for inhibitinglysyl oxidases having formula:


18. The compound of claim 17 or its pharmaceutically acceptable saltadministered in a therapeutically effective amount to treat aneurodegenerative disease, an angiogenesis-related disease, Alzheimer'sdisease, fibrosis including: liver fibrosis, pulmonary fibrosis, renalfibrosis, myocardial fibrosis, cystic fibrosis, idiopathic pulmonaryfibrosis, radiation-induced fibrosis, ocular fibrosis, Peylony's diseaseand scleroderma, or respiratory disorders, abnormal wound healing andrepair, postoperative surgery, cardiac arrest-related fibrosis, excessor abnormal deposition of fibrotic material, all associated withdisorders such as Crohn's disease and inflammatory bowel disease, liver,kidney and pancreas fibrosis, diabetes, cerebral hemorrhage withamyloidosis, cardiac hypertrophy, Hutchinson-Gilford Progeria Syndrome,retinopathy, chemoresistance, and/or a kidney disorder including: kidneyfibrosis, renal fibrosis, acute kidney injury, chronic kidney disease,diabetic nephropathy, glomerulosclerosis, vesicoureteral reflux,tubulointerstitial renal fibrosis and/or glomerulonephritis.
 19. Thecompound of claim 17 or its pharmaceutically acceptable saltadministered as a single agent or in combination with chemotherapy,immunotherapy and/or radiotherapy in both adjuvant and neo-adjuvantsettings.
 20. The compound of claim 17 or its pharmaceuticallyacceptable salt administered with a second therapeutic agent selectedfrom an anti-cancer agent, an anti-inflammatory agent, ananti-hypertensive agent, an anti-fibrotic agent, an anti-angiogenicagent, and/or an immunosuppressive agent.